An Antibody Targeted to VEGFR-2 Ig Domains 4-7 Inhibits VEGFR-2 Activation and VEGFR-2–Dependent Angiogenesis without Affecting Ligand Binding

Kendrew, Jane; Eberlein, Cath; Hedberg, Brad; McDaid, Karen; Smith, Neil R.; Weir, Hazel M.; Wedge, Stephen R.; Blakey, David C.; Foltz, Ian N.; Zhou, Joe Q.; Kang, Jaspal S.; Barry, Simon T.

doi:10.1158/1535-7163.mct-10-0876

Cited by 30 publications

(28 citation statements)

References 33 publications

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“…However, as is evident from previous studies, VEGFR and type III RTK activation requires additional specific homotypic interactions between the membraneproximal domains (22)(23)(24)(25)(26)38). Antibodies blocking these homotypic interactions are promising tools for therapeutic modulation of VEGFR activity (8,26,27). The experiments reported herein introduce a mechanism for VEGFR dimerization and activation via D5 and explain the efficacy of the VEGFR-3 D5-targeted antibodies (8).…”

Section: Discussionsupporting

confidence: 54%

“…We recently reported an antibody against VEGFR-3 D5 that did not block ligand binding but did inhibit VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation and signal transduction (8). Similarly, an antibody and designed ankyrin repeat proteins (DARPins) targeted against D4-7 of VEGFR-2 were found to inhibit ligand-induced receptor activation, but not dimerization (26,27), suggesting that such binders represent a new generation of highly specific inhibitors of VEGFR signaling.In this study, we provide the structural basis of ligand binding to D1-2 of VEGFR-3 and define a unique role of D4-5 for VEGFR dimerization and activation. Using receptor mutants, we show that homotypic interactions in D5 and D7 are essential for VEGFR-3 activation.…”

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confidence: 99%

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Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

Leppänen

Tvorogov

Kisko

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.signal transduction | receptor tyrosine kinase V EGFs stimulate angiogenesis and lymphangiogenesis via VEGF receptors (VEGFRs) in endothelial cells. VEGF-A signaling is mediated predominantly through activation of VEGFR-2, resulting in sprouting of blood vessels from preexisting vasculature (1). In contrast, VEGFR-1 seems to have an inhibitory role by sequestering VEGF-A and thereby preventing its interaction with VEGFR-2 (2). On the other hand, VEGFR-3 plays an indispensable role in lymphangiogenesis (3). VEGFRs are involved in various pathological conditions, including solid tumor growth, tumor metastasis, and vascular retinopathies (4, 5).VEGF-C and VEGF-D compose a VEGFR-3-specific subfamily of VEGFs. They are produced with large N-and C-terminal propeptides and gain activity toward VEGFR-3 and VEGFR-2 on proteolytic processing (reviewed in ref. 5). VEGFR-3 maturation involves proteolytic cleavage of the extracellular domain (ECD) in D5 (6-8). Both VEGF-C and VEGFR-3 also interact with the coreceptor neuropilin-2 (9). Loss of the Vegfc gene results in embryonic lethality owing to a lack of lymphatic vessel formation (10), whereas mutations that interfere with VEGFR-3 signaling have been associated with hereditary lymphedema, and mice deficient in Vegfr3 die in utero due to abnormal development of the blood vasculature (11, 12). VEGFR-3 and its heterodimers with VEGFR-2 are also important for sprouting angiogenesis and vascular network formation (13-15).VEGFRs are type V rece...

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Section: Discussionsupporting

confidence: 54%

mentioning

confidence: 99%

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confidence: 99%

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Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

Leppänen

Tvorogov

Kisko

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…For both assays, antibody titrations were prepared in MCDB131 media supplemented with 2% FCS. Details of the methods are described in the work of Kendrew and colleagues (22) and shown in the Supplementary Materials and Methods.…”

Section: Two-dimensional Fibroblast-endothelial Cells Coculture Assaysmentioning

confidence: 99%

“…This assay was run as previously described (22). Detailed method is shown in the Supplementary Materials and Methods.…”

Section: Three-dimensional Tubule Formation Assaymentioning

confidence: 99%

MEDI0639: A Novel Therapeutic Antibody Targeting Dll4 Modulates Endothelial Cell Function and Angiogenesis In Vivo

Jenkins

Ross

Veldman-Jones

et al. 2012

Molecular Cancer Therapeutics

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The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand-receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody crossreacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell-fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway.

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Phthalazine‐based VEGFR‐2 inhibitors: Rationale, design, synthesis, in silico, ADMET profile, docking, and anticancer evaluations

Khedr

Ibrahim

Eissa

et al. 2021

Archiv der Pharmazie

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In the designed compounds, a new linker was inserted in the form of fragments with verified VEGFR‐2 inhibitory potential, including an α,β‐unsaturated ketonic fragment, pyrazole, and pyrimidine. Also, new distal hydrophobic moieties were attached to these linkers that are expected to increase the hydrophobic interaction with VEGFR‐2 and, consequently, the affinity. These structural optimizations have led us to identify the novel dihydropyrazole derivative 6e as a promising hit molecule. All the new derivatives were evaluated to assess their anticancer activity against three human cancer cell lines, including HepG2, HCT‐116, and MCF‐7. The results of the in vitro anticancer evaluation study revealed the moderate to excellent cytotoxicity of 6c, 6e, 6g, and 7b, with IC50 values in the low micromolar range. The inhibitory activity of VEGFR‐2 was investigated for 16 of the designed compounds. The enzyme assay results of the new compounds were compared with those of sorafenib as a reference VEGFR‐2 inhibitor. The obtained results demonstrated that our derivatives are potent VEGFR‐2 inhibitors. The most potent derivatives 6c, 6e, 6g, and 7b showed IC50 values in the range of 0.11–0.22 µM. Molecular docking and pharmacokinetic studies were also conducted to rationalize the VEGFR‐2 inhibitory activity and to evaluate the ability of the most potent derivatives to be developed as good drug candidates.

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An Antibody Targeted to VEGFR-2 Ig Domains 4-7 Inhibits VEGFR-2 Activation and VEGFR-2–Dependent Angiogenesis without Affecting Ligand Binding

Cited by 30 publications

References 33 publications

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

MEDI0639: A Novel Therapeutic Antibody Targeting Dll4 Modulates Endothelial Cell Function and Angiogenesis In Vivo

Phthalazine‐based VEGFR‐2 inhibitors: Rationale, design, synthesis, in silico, ADMET profile, docking, and anticancer evaluations

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