VEGF Binding Research Articles

A conformation-based phage-display panning to screen neutralizing anti-VEGF VHHs with VEGFR2 mimicry behavior

The potency of VEGF-based anti-angiogenic strategies in cancer therapy and the brilliant characteristics of VHHs motivated us to directly block VEGF binding to its receptor with neutralizing single domain antibodies, thereby fading away the VEGF signaling pathway. Considering with high resolution crystal structure of VEGF-RBD/VEGFR2 complex, we could adopt a combinatorial screening strategy: stringent panning and competition ELISA, to direct the panning procedure to dominantly screen the favorable binders that bind and block the key functional regions of VEGF. Based on competition assay, the majority of the screened clones (82%) showed the VEGFR2 mimicry behavior for binding to VEGF molecule. The phage pool gets enriched in favor of sequences that bind the receptor binding sites of VEGF. Different immunoassays and molecular docking simulation verified that all selected VHHs could bind and cover the receptor binding sites of VEGF. Consequently, some modifications in panning procedure with considering the structural features and detailed information of functional regions of a protein antigen, led us to successfully trap the high-affinity specific binders against its hot functional regions. Since the selected VHHs could cover the receptor binding site of VEGF and block VEGF binding to the receptor, they might be promising candidates for anti-angiogenic therapies.

Binding of VEGF-A is sufficient to abrogate the disturbing effects of VEGF-B together with VEGF-A on retinal endothelial cells.

PurposeInhibition of vascular endothelial growth factor (VEGF) is a promising strategy to treat retinal complications of diabetes. In contrast to VEGF-A binding ranibizumab, aflibercept also binds to other members of the VEGF family including VEGF-B, but potential effects of this factor on permeability and angiogenic processes are unclear. Therefore, we studied how VEGF-B variants as single agents or together with VEGF-A165 might affect proliferation, migration, or barrier function of retinal endothelial cells (REC). Also investigated was the normalization of REC properties with both VEGF-inhibitors to explore if additional targeting of VEGF-B is relevant.MethodsStimulation of proliferation or migration of immortalized bovine REC (iBREC) and disturbance of their barrier by exposure to VEGF-B variants (as single factors or together with VEGF-A165) was determined with or without VEGF-binding proteins being added. Permeability of iBREC was assessed by measuring their transendothelial resistance (TER) and expression of the tight junction protein claudin-1.ResultsVEGF-B167 and VEGF-B186 enhanced proliferation of iBREC but these isoforms did not affect cell migration. Interestingly, ranibizumab completely blocked both migration and proliferation induced by VEGF-A plus VEGF-B. Both VEGF-B variants did also not affect barrier function or claudin-1 expression in a normal or high-glucose environment. Accordingly, binding VEGF-A was enough to normalize a reduced TER and reinstate claudin-1 lost during treatment with this factor in combination with VEGF-B.ConclusionsImportant properties and functions of REC seem not to be affected by any VEGF-B variant and targeting the key factor VEGF-A is sufficient to normalize growth factor-disturbed cells of this type.Electronic supplementary materialThe online version of this article (doi:10.1007/s00417-015-2944-z) contains supplementary material, which is available to authorized users.

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Objectives We evaluated role of sFlt-1 and VEGF in a preeclampsia model driven by RAS activation. We hypothesized as transgenic crossbreeds develop preeclampsia, features of hypertension, proteinuria, kidney and placenta end-organ damage will occur concurrently with (1) sFlt1 rising higher in preeclamptic mice, (2) VEGF remaining high in preeclamptic mice but equivalent to controls; and, (3) preeclamptic kidneys demonstrating increased VEGF binding. Methods Transgenic hAGT and hREN, or wild type (WT) mice were used. Non-gravid females underwent telemetric transmitter implantation for blood pressure (BP) measurements. Mice were cross-bred: hAGT × hREN for RAS preeclamptic model and pregnant controls (WT × WT). Blood and urine collected on days (GD) 12, 15, and 18. Vascular reactivity in mesenteric arteries investigated by wire myography using KCl, acetylcholine (ACH), phenylephrine (PE). ELISA performed on urine for albumin and on plasma for VEGF and sFlt-1. Necropsies were performed GD 18-19. Kidneys and placenta were sectioned for HE and immunostain. Results 11 RAS pregnancies and 9 WT pregnancies (WTP) compared. 12,414 h of BP data analyzed. RAS mice demonstrated higher BP and proteinuria. RAS mice had glomerular endotheliosis (80% vs. 11%; p = 0.02), and placental necrosis (60% vs. 0%; p Conclusions While RAS model of preeclampsia recapitulates human preeclamptic state with high fidelity, renal injury may actually be mediated by increased VEGF binding. Disclosures J.M. Denney: None. C.E. Shaw: None. A. Gendron-Fitzpatrick: None. I. Bird: None. D. Shah: None.

Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity.

Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.

Ethoxyfagaronine, a synthetic analogue of fagaronine that inhibits vascular endothelial growth factor-1, as a new anti-angiogeneic agent.

Angiogenesis plays a pivotal role in tumorigenesis and also contributes to the pathogenesis of hematologic malignancies. A number of plant compounds have shown efficacy in preclinical and clinical studies and some of them possess an anti-angiogenic activity. Our present findings report anti-angiogenic activities of ethoxyfagaronine (etxfag), a synthetic derivative of fagaronine. Once determined the non-cytotoxic concentration of etxfag, we showed that the drug inhibits VEGF-induced angiogenesis in a Matrigel™ plug assay and suppresses ex vivo sprouting from VEGF-treated aortic rings. Each feature leading to neovascularization was then investigated and results demonstrate that etxfag prevents VEGF-induced migration and tube formation in human umbilical vein endothelial cells (HUVEC). Moreover, etxfag also suppresses VEGF-induced VEGFR-2 phosphorylation and inhibits FAK phosphorylation at Y-861 as well as focal adhesion complex turnover. Beside these effects, etxfag modifies MT1-MMP localization at the endothelial cell membrane. Finally, immunoprecipitation assay revealed that etxfag decreases VEGF binding to VEGFR-2. As we previously reported that etxfag is able to prevent leukemic cell invasiveness and adhesion to fibronectin, all together our data collectively support the anti-angiogenic activities of etxfag which could represent an additional approach to current anti-cancer therapies.

An immunodepletion procedure advances free angiopoietin-2 determination in human plasma samples during anti-cancer therapy with bispecific anti-Ang2/VEGF CrossMab

Bispecific monoclonal IgG antibodies offer increased efficacy by antagonizing two different targets. Assessing drug mechanisms, target engagement and biomarker features, the quantification of free target levels is essential. The anti-Ang2/VEGF-CrossMab (anti-A2V) recognizes soluble vascular endothelial growth factor-A (VEGF-A) and soluble angiopoietin-2 (Ang2). However, an assay for reliable free Ang2 determination is missing. Here, we describe an immunodepletion procedure that allows for selective quantification of free Ang2 target levels by taking into advantage the bispecificity of the therapeutic antibody. The specificity for VEGF was utilized to efficiently eliminate drug-bound Ang2 from plasma samples prior to an established Ang2 measurement. The magnetic bead-based depletion procedure used an anti-idiotypic monoclonal antibody (mAb) specific for the VEGF binding site of anti-A2V (anti-Id-anti-VEGF mAb) to capture the drug along with drug-bound Ang2. High efficiencies of 99.9% were obtained for anti-A2V depletion (concentration range 300ng/mL to 106ng/mL) reflecting a 1000-fold reduction of drug-bound Ang2. A significant impact of the interaction of anti-Id-anti-VEGF mAb with anti-A2V on the Ang2 binding could be excluded. Moreover, reliable quantification of free Ang2 concentrations in plasma samples was assured by interference testing. Performing advanced free Ang2 determination including the immunodepletion step in parallel to established Ang2 measurement without immunodepletion, we compared free with total Ang2 concentrations in human plasma samples obtained from an anti-A2V Phase 1 clinical study. Samples from untreated patients displayed rather low and equal values for both free and total Ang2. In contrast, samples from drug-treated patients showed a significant reduction of free Ang2 accompanied by an accumulation in total Ang2. These results underline the value of the novel immunodepletion procedure for reliable discrimination of free vs. total target quantification with particular importance for pre-clinical and clinical development of anti-A2V. Moreover, this approach may serve as universal concept for the determination of free target levels of bispecific therapeutic antibodies.

Abstract 2600: Preclinical evaluation of the novel fusion molecule with high dual antiangiogenic and anticancer potential

Abstract Angiogenesis, the process of new blood vessels formation, is essential for tumor growth and metastasis. Since tumors are unable to grow beyond a millimeter diameter in the absence of angiogenic factors, it was postulated that blocking of angiogenic factors could provide a new strategy to inhibit tumor leading to a potential novel anticancer therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) was considered as a promising anticancer agent due its remarkable ability to induce apoptosis in cancer cells without harming normal cells. However, in clinical practice TRAIL efficacy was too low to become the effective single therapy agent. Here we present a novel fusion protein based on TRAIL/Apo2L and equipped with an antiangiogenic effector peptide originating from VEGF exon 6 that binds to natural VEGF receptors and competes with their natural ligand. The proposed fusion protein, AD-O51.4, consists of extracellular soluble portion of TRAIL linked to two tandemly arranged copies of the effector peptide, separated by a sequence that contains the motif recognized by tumor-specific proteases (MMP's, uPa). The AD-O51.4 fusion molecule exerts its dual cytotoxic and antiangiogenic activity upon interaction with cellular death and VEGF receptors on cancer endothelial cells. In consequence, the peptide blocks the binding of VEGF to its receptors, preventing new vessels formation. In addition, due to the presence of VEGF receptors on cancer cells, they can be also targeted by VEGF receptors-binding domain of AD-O51.4 making them susceptible for TRAIL-induced apoptosis. Indeed, an extensive panel of discovery-stage studies of AD-O51.4 showed broad cytotoxic activity on primary cancer cell lines but no toxic effects on normal cells and strong antitumor activity on subcutaneous and orthotropic xenograft models with complete tumor remission. Based on these data, early preclinical part of the program was initiated. An LC\MS-based method, specific for the intact molecule, was set up as the detection and quantification tool, enabling detection of AD-O51.4 in different biological matrices. Safety of the molecule was evaluated using healthy hepatocytes form humans and cynomolgus monkeys. To verify tumor targeting capacity of the molecule, tumor-specific accumulation in subcutaneous xenograft model of human colorectal cancer as a function of time was assessed using imaging techniques. Tumor specific accumulation was also confirmed using LC\MS method even after single administration. To analyse the pharmacological properties of the AD-O51.4, PK profiles were evaluated in Wistar rats and cynomoglus monkeys. Finally, strong antitumor activity was confirmed using PDX models of human lung, colon, rectal and pancreatic cancer. Currently the molecule is approaching formal ADME\Tox panel. Citation Format: Jerzy S. Pieczykolan, Anna Pieczykolan, Piotr Rozga, Sebastian Pawlak, Bartlomiej Zerek, Malgorzata Teska - Kaminska, Szymanik Michal, Albert Jaworski, Karl R. Erlemann, Jacek Capala. Preclinical evaluation of the novel fusion molecule with high dual antiangiogenic and anticancer potential. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2600. doi:10.1158/1538-7445.AM2014-2600

Axitinib Improves Radiotherapy in Murine Xenograft Lung Tumors

A third of patients with non-small cell lung cancer (NSCLC) present with un-resectable stage III locally advanced disease and are currently treated by chemo-radiotherapy but the median survival is only about 21months. Using an orthotopic xenograft model of lung carcinoma, we have investigated the combination of radiotherapy with the anti-angiogenic drug axitinib (AG-013736, Pfizer), which is a small molecule receptor tyrosine kinase inhibitor that selectively targets the signal transduction induced by VEGF binding to VEGFR receptors. We have tested the combination of axitinib with radiotherapy in nude mice bearing human NSCLC A549 lung tumors. The therapy effect was quantitatively evaluated in lung tumor nodules. The modulation of radiation-induced pneumonitis, vascular damage and fibrosis by axitinib was assessed in lung tissue. Lung irradiation combined with long-term axitinib treatment was safe resulting in minimal weight loss and no vascular injury in heart, liver and kidney tissues. A significant decrease in the size of lung tumor nodules was observed with either axitinib or radiation, associated with a decrease in Ki-67 staining and a heavy infiltration of inflammatory cells in tumor nodules. The lungs of mice treated with radiation and axitinib showed a complete response with no detectable residual tumor nodules. A decrease in pneumonitis, vascular damage and fibrosis were observed in lung tissues from mice treated with radiation and axitinib. Our studies suggest that axitinib is a potent and safe drug to use in conjunction with radiotherapy for lung cancer that could also act as a radioprotector for lung tissue by reducing pneumonitis and fibrosis.

N-terminal modification of VEGF-A C terminus-derived peptides delineates structural features involved in neuropilin-1 binding and functional activity.

The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.

Tissue Inhibitor of Metalloproteinase-3 Knockout Mice Exhibit Enhanced Energy Expenditure through Thermogenesis

Tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme), its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO) mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT) mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.

Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells

Members of the vascular endothelial growth factor (VEGF) family differently regulate processes in retinal endothelial cells (REC) which are crucially involved in the pathogenesis of diabetic retinopathy: Both, VEGF-A and placenta growth factor (PlGF), stimulate proliferation of primary and immortalized bovine REC ((i)BREC) but only VEGF-A165 stimulates their migration. Diabetic macular edema is most likely a consequence of an elevated permeability of REC which can be induced by VEGF-A, but not by PlGF. Binding of VEGF-A by the antibody fragment ranibizumab is sufficient to completely restore or prevent VEGF-A-induced disturbance of the iBREC barrier or migration of these cells without affecting the basal processes. This was observed even in the presence of other growth factors when surplus proliferation was only partly blocked. The recombinant protein aflibercept (VEGF-trap) not only binds very strongly to VEGF-A, but – in contrast to ranibizumab – also recognizes PlGF. In this study, we investigated whether this additional targeting of PlGF also results in better inhibition of growth factor-induced proliferation and migration, and disturbance of the iBREC barrier. In addition, uptake of aflibercept by iBREC and potential functional consequences were examined. In accordance with its binding specificity, aflibercept strongly and specifically inhibited iBREC proliferation stimulated with VEGF-A, PlGF or a combination of these factors. By treatment with aflibercept at therapeutically achievable concentrations, VEGF-A-stimulated iBREC migration was reduced not only to normal values but driven below the basal level. However, the VEGF-A binding humanized antibody bevacizumab as well as the unrelated control antibody rituximab also inhibited basal or VEGF-A stimulated migration at clinically relevant concentrations, suggesting an effect of high amounts of IgG domain-containing proteins which does not depend on their binding specificity. However, aflibercept specifically blocked VEGF-A stimulated migration at lower concentration without influencing basal processes. Effects on permeability were determined by measuring transendothelial resistance (TER) of iBREC (±VEGF-A165) and their expression of the tight junction protein claudin-1. The VEGF-A-disturbed barrier was completely restored by treatment with ≤25 μg/ml aflibercept of which even much higher concentrations did not interfere with normal barrier function. Uptake of aflibercept by iBREC – analyzed by Western blot – was observed after 1 h of treatment and the amount further increased during prolonged incubation. Most of the internalized aflibercept was present in subcellular fractions of proteins assigned to the membranes and organelles, but it was also detected in the fraction consisting of cytoskeletal proteins. Co-immunofluorescence staining showed aflibercept absorbed by iBREC to be localized in or close to the Golgi apparatus. Aflibercept at high concentrations interferes with an important normal iBREC function, but prevents and restores VEGF-A-induced disturbances at considerably lower concentrations. Therefore, reduction of the doses administered in DR and DME therapy might be considered.

A phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors

The human monoclonal antibody MNRP1685A targets the VEGF binding domain of neuropilin-1 (NRP1), a multi-domain receptor necessary for neural development and blood vessel maturation. In nonclinical studies, MNRP1685A prevents vascular maturation by keeping blood vessels in an immature, highly VEGF-dependent state. We explored the safety and tolerability of MNRP1685A in patients with advanced solid tumors. Patients were treated with MNRP1685A given intravenously every 3 weeks using a 3 + 3 dose-escalation design with 7 dose-escalation cohorts. Twenty-four of 35 patients (69%) experienced drug-related adverse events (AEs) of infusion-related reaction on the day of MNRP1685A administration. With premedication including dexamethasone, infusions were well-tolerated with main symptoms of pruritus and rash. Outside the day of infusion, most common (≥ 2 patients) related AEs were fatigue (17%), pruritus (9%), myalgia and thrombocytopenia (both 6%) (all were Grade 1-2). MNRP1685A-related Grade ≥ 3 AEs consisted of one dose-limiting toxicity of Grade 3 upper gastrointestinal bleeding and one related Grade 3 thrombocytopenia, coinciding with unrelated Grade 3 fungemia and duodenal obstruction. MNRP1685A showed nonlinear PK with more-than-dose proportional increases in exposure, consistent with broad target expression. Transient platelet count reductions (≥ 30% from predose) were observed in 56% of evaluable patients. Nine patients were on study for ≥ 4cycles, one colorectal cancer patient for one year. MNRP1685A was generally well-tolerated. The primary MNRP1685A-related AE was infusion-related reaction, which were attenuated by premedication including dexamethasone. Transient platelet count reductions were frequent but did not impact MNRP1685A dosing.

A1.36 Active immunisation against peptides of vegf improves joint inflammation and destruction in collagen-induced arthritis

BackgroundAngiogenesis is a key pathophysiological process in rheumatoid arthritis (RA). VEGF-A is considered the major player in both physiological and pathological angiogenesis. VEGF–VEGFR system blockade delays synovitis onset and ameliorates...

Neuropilin 1 (NRP1) hypomorphism combined with defective VEGF-A binding reveals novel roles for NRP1 in developmental and pathological angiogenesis

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth.

Binding of VEGF-A to canine cancer cells with preferential expression of VEGFR1

Aim: Despite encouraging results in syngeneic and xenografts cancer models with various inhibitors of vascular endothelial growth factor (VEGF) or its receptors (VEGFRs), beneficial effects have not been consistently translated to the clinic, underscoring the need to develop strategies that go beyond the inhibition of these targets. The purpose of this study was to generate data to support the hypothesis that VEGF may be used as “bait” to selectively deliver therapeutics to VEGFRexpressing cancer cells. Materials and Methods: VEGFR1 and VEGFR2 expression was characterized using real time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in canine hemangiosarcoma (Grace-HSA, Emma-HSA), melanoma (TLM-1), and thyroid adenocarcinoma (CTAC) cell lines. TLM-1 and Grace-HSA were identified as representative cell lines that selectively expressed high levels of VEGFR1. Flow cytometry was performed to examine binding of a single VEGF molecule (biotinylated VEGFA and avidin conjugated to fluorescein isothiocyanate (FITC)) by these chemoresistant cell

Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling

Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.

Mechanistic Basis for the Potent Anti-Angiogenic Activity of Semaphorin 3F

Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.

Effects of Semaphorin 3A on Retinal Pigment Epithelial Cell Activity

Semaphorin 3A (Sema3A), a chemorepellant guidance protein, has been shown to be crucial for neural and vascular remodeling. This study is designed to examine the effects of Sema3A on RPE cell activity both in vitro and in vivo. Retinal pigment epithelial were incubated with Sema3A, or VEGF- and Sema3A-containing medium. Cell proliferation, migration, cell cycle, apoptosis, cocultured human umbilical vein endothelial cells tube formation, VEGF receptor 2 (VEGFR2) and neuropilin 1 (Nrp1) receptor expression, VEGF- and pigment epithelium-derived factor (PEDF) concentration, and c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38MAPK) signaling pathway studies were measured. A rabbit proliferative vitreoretinopathy (PVR) model was used for in vivo study. Subconfluent ARPE19 cells were injected intravitreously with or without Sema3A. Data were analyzed with Graphpad Prism 5.0 software. In vitro, Sema3A not only induced RPE cell cycle arrest and inhibited RPE migration under normal culture conditions, but also inhibited exogenous and endogenous VEGF165-induced cell activities. These activities included proliferation, migration, cell cycle arrest, JNK and p38MAPK signaling pathway phosphorylation, and cocultured endothelial cell tube formation. It is shown that both VEGF165 and Sema3A induced the upregulation of VEGFR2 and Nrp1 receptors. Activity inhibition was mediated by impeding VEGF165 utilization and possibly mediated by competitive inhibition of VEGF165 binding to its receptor VEGFR2, but not by the suppression of VEGF165 secretion. In vivo, Sema3A inhibited PVR, which is induced by RPE proliferation. These results suggested that Sema3A could be a useful therapeutic strategy for preventing RPE malfunction.

The protective effect of albumin on bevacizumab activity and stability in PLGA nanoparticles intended for retinal and choroidal neovascularization treatments

The rapidly growing applications of antibody-based therapeutics requires novel approaches to develop efficient drug delivery systems in which biodegradable polymeric nanoparticles are amongst the best candidates. In the present study bevacizumab loaded PLGA nanoparticles were formulated by water-in-oil-in-water emulsion method. Protein inactivation and aggregation are the major drawbacks of this technique. Therefore protective ability of various stabilizers was studied during entrapment process. Probable changes in VEGF165 binding capability of bevacizumab was assayed by ELISA which portrays the antibody’s bio-efficiency. Probable breakage of bevacizumab and its secondary and tertiary structural integrity upon entrapment were analyzed by SDS–PAGE and circular dichroism spectroscopy, respectively. In vitro and ex vivo released bevacizumab from the prepared nanoparticles was also investigated. Results revealed that the protein interfacial adsorption is the foremost destabilizing factor in the double emulsion method and incorporation of appropriate concentrations of albumin could protect bevacizumab against entrapment stress. Ex vivo release results, in rabbit vitreous, indicated the ability of prepared nanoparticles in prolonged release of the active antibody. Consequently this approach was an attempt to achieve sustained release PLGA nanoparticle formulation with the aim of protecting integrity and performance of entrapped bevacizumab.

Abstract 4544: Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors.

Abstract Vascular endothelial growth factor (VEGF) is potent mitogen for endothelial cells that signals through VEGF Receptor-type2 (VEGFR-2)/Flk-1/KDR. Binding of VEGF to VEGFR-2, a membrane bound receptor tyrosine kinase of the VEGF-receptor family, leads to receptor dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling pathways stimulating angiogenesis. In adults, angiogenesis normally occurs during wound healing to restore blood flow post injury and during menstruation and pregnancy. Angiogenesis is also involved in several pathological disease states such as rheumatoid arthritis, psoriasis, diabetic retinopathy and cancer. While physiological angiogenesis involves interplay between pro- and anti-angiogenic factors, the ability to inhibit pathological angiogenesis has therapeutic potential. Inhibitors of VEGFR-2 provide targeted treatments for tumor growth as well as abnormal neovascularization related diseases. The caveat of traditional biochemical VEGFR-2 assays is that only the catalytic domain of the receptor is used to identify hits. One strategy to identify true novel inhibitors against membrane associated proteins is to screen these targets in a more physiological environment, where lipid-protein and protein-protein interactions are included in the assay. We achieved this goal by docking the entire cytoplasmic domain of the receptor on a liposome. Template Directed Assembly (TDA) is a screening technology designed to accomplish this objective. TDA technology utilizes recombinant VEGFR-2 cytoplasmic domain tagged with polyhistidine at the juxtamembrane position and docked to the liposome via the nickel residues. The lipids in TDA are able to diffuse within the surface of the liposome and, therefore, proteins can diffuse to form oligomeric complexes such as homo- or hetero-dimers formation. To compliment the TDA biochemical screen, we are implementing an image based high content screen (HCS) utilizing human umbilical vein endothelial cells (HUVECs) that express native VEGFR-2. The HCS screen is designed to identify potent and specific inhibitors of VEGFR-2 as assessed by a reduction in the proliferation marker Ki67. We are currently (1) validating the TDA assay with known VEGFR-2 inhibitors (2) screening a focus small molecule library to identify novel hits (3) testing the primary hits against a kinase selectivity panel and (4) designing and developing a VEGFR2 cell-based HCS assay to confirm the primary hits. The hit to lead program will be guided by using both physiologically relevant biochemical-based and cell-based assays. The combination of our novel TDA screening platform and the HCS assay technology permits us to identify unique VEGFR-2 inhibitors. Citation Format: Kelvin Lam, Edward Esposito, Deanna Navaroli, Ryan Clinton, Kerrin Beovich, Norman Garceau. Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2013-4544