Vascular Endothelial Growth Factor Antibody Research Articles

Vascular Endothelial Growth Factor and Spinal Cord Injury Pain

Vascular endothelial growth factor (VEGF)-A mRNA was previously identified as one of the significantly upregulated transcripts in spinal cord injured tissue from adult rats that developed allodynia. To characterize the role of VEGF-A in the development of pain in spinal cord injury (SCI), we analyzed mechanical allodynia in SCI rats that were treated with either vehicle, VEGF-A isoform 165 (VEGF(165)), or neutralizing VEGF(165)-specific antibody. We have observed that exogenous administration of VEGF(165) increased both the number of SCI rats that develop persistent mechanical allodynia, and the level of hypersensitivity to mechanical stimuli. Our analysis identified excessive and aberrant growth of myelinated axons in dorsal horns and dorsal columns of chronically injured spinal cords as possible mechanisms for both SCI pain and VEGF(165)-induced amplification of SCI pain, suggesting that elevated endogenous VEGF(165) may have a role in the development of allodynia after SCI. However, the neutralizing VEGF(165) antibody showed no effect on allodynia or axonal sprouting after SCI. It is possible that another endogenous VEGF isoform activates the same signaling pathway as the exogenously-administered 165 isoform and contributes to SCI pain. Our transcriptional analysis revealed that endogenous VEGF(188) is likely to be the isoform involved in the development of allodynia after SCI. To the best of our knowledge, this is the first study to suggest a possible link between VEGF, nonspecific sprouting of myelinated axons, and mechanical allodynia following SCI.

Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy

Background:The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC.Methods:Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification.Results:Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31% P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05).Conclusion:Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.

In Vitro Effects of Antivascular Endothelial Growth Factors on Cultured Human Trabecular Meshwork Cells

To investigate the effects of a neutralizing vascular endothelial growth factor antibody and antibody fragment as well as vehicle components on primary cultures of human trabecular meshwork (TM) cells. Assays of cellular metabolism were performed using the diphenyl tetrazolium bromide assay in confluent cultures of cells. Proliferative effects were determined by measuring 5'-bromo-2'-deoxyuridine uptake in subconfluent cultures of cells. Twenty-four-hour treatment with 4 mg/mL bevacizumab reduced TM metabolism to 34.4 ± 12.4% (mean ± SD) as compared with human immunoglobulin G controls (P<0.0001). 4 mg/mL bevacizumab also reduced TM cell proliferation to 62.7 ± 9.2% of controls (P<0.0001). No significant decrease was seen at 2 mg/mL bevacizumab, or with molar equivalents of the related anti-vascular endothelial growth factor agent ranibizumab. Exposure of TM cells to the components of bevacizumab and ranibizumab vehicle did not lead to significant antimetabolic effects. Our data reveal that high concentrations of bevacizumab are harmful to TM cells in vitro whereas no such effect was noted with human immunoglobulin G controls or ranibizumab. Further studies are needed to better understand the antimetabolic effects of higher concentrations of bevacizumab on intraocular cell lines and whether smaller concentrations may have a similar effect on TM cells after repeated exposures.

Prolongation of Islet Graft Survival Using Concomitant Transplantation of Islets and Vascular Endothelial Cells in Diabetic Rats

Background The transplantation of isolated islets of Langerhans is nearing acceptance as treatment of type 1 diabetes mellitus. Because the arterial and venous connections of the pancreas are disrupted during islet isolation, islets must be revascularized after transplantation. Objective To observe whether increased numbers of vascular endothelial cells in islets can affect the angiogenesis and function of the grafts. Materials and Methods Rats with streptozocin-induced diabetes were divided into 3 groups. The rats in group 1 received islet grafts under the capsule of the left kidney; rats in group 2 received combined vascular endothelial cell and islet transplants; and rats in group 3 served as controls. After the transplantation procedure, blood glucose and insulin concentrations were evaluated daily. Hematoxylin-eosin and immunohistochemical staining was used to detect expression of vascular endothelial growth factor antibodies in the diabetic rat kidneys. The mean microvascular density was also calculated. Results At 3 days posttransplantation, blood glucose and insulin concentrations returned to normal in group 2, however, they declined only slightly in group 1, and moderate hyperglycemia was present. There was a significant difference in blood glucose and insulin concentrations between the 2 groups after 3 days ( P < .05). The mean (SD) microvascular density in group 2 was markedly higher than that in group 1 (12.58 [1.81] vs 10.38 [0.97] P = .04). Conclusion This study suggests that concomitant transplantation of isolated islets with endothelial cells can prolong islet graft survival in diabetic rats.

VEGF-Antikörper in der Therapie der Frühgeborenenretinopathie

Retinopathy of prematurity (ROP) is one of the three leading causes of legal blindness in childhood in the developed countries. Improved neonatal care has resulted in the increased survival of extremely immature infants at high risk to develop ROP. Current treatment for ROP with laser may prevent blindness by causing involution of pathological vessels and thus inhibit the development of retinal detachment. But this coagulation of the avascular retina is a destructive therapy and does not otherwise ameliorate retinal development. Recent reports have described vascular endothelial growth factor antibodies as therapy for ROP. This article reports our own experience with this new therapy and gives an overview of the recent literature.

Die Rolle der Anti-VEGF-Therapie in der Behandlung des diabetischen Makulaödems

Diabetic macular edema (DME) and proliferative retinopathy are common causes for blindness in middle-aged patients. The gold standard for treatment has been laser coagulation. Limitations of this therapy are refractive DME, ischaemic diabetic maculopathy and complications after laser application. The need for a non-destructive and effective strategy has led to investigations regarding vascular endothelial growth factor (VEGF) inhibitors for reduction of vessel leakage and oedema formation. VEGF antibody fragments have been approved for age-associated macular degeneration (AMD) in different countries and have proved to be safe and effective. Trials for different anti-VEGF compounds are nearing completion or are completed. Published data show a superiority of anti-VEGF therapy compared to laser coagulation. In the present article, data regarding dose, injection scheme and practicable application form are reviewed.

Praziquantel efficacy in mice infected with PZQ non‐susceptible S. mansoni isolate treated with artemether: parasitological, biochemical and immunohistochemical assessment

Based on the fact that artemether (ART) affects immature schistosomes and that the effect of praziquantel (PZQ) mainly targets mature schistosomes, this work investigates the possible enhanced efficacy of PZQ in combination with ART in mice harboring a PZQ non-susceptible Schistosoma mansoni isolate. Associated schistosomal, inflammatory, hepatic histopathological changes have been investigated by examining the tissue markers expressing apoptosis using FAS (CD95), anti-apoptosis (Bcl2) and angiogenesis [vascular endothelial growth factor (VEGF)]. A batch of Swiss albino mice infected with a PZQ non-susceptible (EE10) S. mansoni isolate was divided into 12 groups. Animals of the first group were left without treatment as infected controls, while groups 2-6 received PZQ in increasing doses. The animals of group 7 received ART in double doses. Those comprising groups 8-12 received combined therapy of PZQ and ART in the same doses and at the same timings postinfection (PI) as those belonging to groups 2-6. Parasitological parameters, liver function, and histopathological and immunohistochemical studies of FAS, Bcl2 and VEGF antibodies were assessed. Combined administration of ART and PZQ reduced the ED(50) (the dose at which the worm burden was decreased by 50%) of PZQ. Typical granulomas were not seen in animals treated with ART alone and combined with PZQ, with least expression of FAS and VEGF and increased expression of Bcl2. The minimal histopathological changes recorded in mice treated with both ART and PZQ could be related to a synergistic/additive effect of ART, markedly reducing the intensity of infection. Improved liver function tests support the less severe histopathological changes under the influence of this treatment protocol. This study encourages human trials especially in areas where malaria is not endemic, and differing combination doses should be investigated in view of the antagonistic effect noticed with some dose regimens.

What Type of VEGF Do You Need?

Typically considered a mediator of angiogenesis and vascular permeability, vascular endothelial growth factor (VEGF) has emerged as an important modulator of glomerular health and disease. The pleiotropic nature VEGF was dramatically illustrated in cases in which patients who were undergoing chemotherapy with VEGF antibodies ( e.g. , bevacizumab) developed proteinuria1 and thrombotic microangiopathy,2 indicating damage to the glomerular filtration barrier. In addition, individuals with preeclampsia, a condition characterized by excess circulating levels of a soluble form of the VEGF receptor, can likewise develop proteinuria,3 and increased serum levels of sFlt may contribute to endothelial dysfunction in chronic kidney disease.4 These studies suggest that a threshold of VEGF must be met to ensure glomerular health. Accordingly, in this editorial space just 4 years ago, Mathieson asked, “How much VEGF do you need?”5 At the time, the question was prompted by several studies demonstrating that VEGF gene dose is critical to glomerular development, structure, and function. Quaggin's group6,7 was reporting the generation of a series of gene-targeted mice with podocyte-specific VEGF reduction, with expression ranging from 0 to 50% of normal. This exquisite control over VEGF expression yielded a striking variety of phenotypes: When VEGF expression is ablated entirely, glomerular development fails and perinatal lethality ensues; low expression levels (approximately 25% of normal) results in a cascade of glomerular cell attrition beginning with endothelial and proceeding to mesangial cells shortly after birth, and intermediate VEGF levels (approximately 50% of normal) recapitulates …

Imaging of angiogenesis: from morphology to molecules and from bench to bedside

Angiogenesis is one of the hallmarks of cancer and is increasingly in the focus of oncological research. Judah Folkman first articulated the importance of angiogenesis for tumor growth in 1971 [1]. He stated that the growth of solid tumors remains restricted to 2–3 mm in diameter until the onset of angiogenesis. Although this hypothesis first was strongly criticized, angiogenesis soon became one of the most important fields of research in oncology. Up to now, subsequent investigations have identified more than 20 angiogenic growth factors, their receptors and signal transduction pathways. Moreover, endogenous angiogenesis inhibitors have been discovered, and the cellular and molecular characterization of the angiogenic phenotype in human cancers has been achieved [2, 3]. Although the results of the first clinical trials using angiogenesis inhibitors in oncology were disappointing, encouraging results have been achieved in the past few years with the vascular endothelial growth factor (VEGF) antibody Avastin® in combination with standard cytotoxic chemotherapy. This combined antiangiogenic-cytotoxic therapy has been proven to be successful first in metastasized colorectal cancer, and subsequently also in breast cancer and non-small cell lung cancer [4–7]. This success of targeted antiangiogenic therapy will spur the demand for imaging modalities for assessment of the angiogenic cascade, which is the topic of this supplement “Imaging of Angiogenesis.” Imaging of angiogenic activity might on the one hand help in the process of developing novel antiangiogenic drugs in the preclinical setting. Moreover, in the clinical setting, imaging of angiogenesis might be useful for assessment of the optimum dose of new antiangiogenic agents and for early response evaluation. Up to now, clinical trials with conventional cytotoxic chemotherapeutic agents have mainly used morphological imaging to provide indices of therapeutic response, mostly computed tomography (CT) or magnetic resonance imaging (MRI). Bidimensional measurements of the maximum tumor extension are mainly used to estimate changes in response to the investigational therapy as compared with a baseline measure. Through standardization of these measurements by introducing the RECIST criteria in the year 2000, considerable progress has been achieved [8]. However, as antiangiogenic agents lead to a stop of tumor progression rather than to tumor shrinkage, the approach of measuring tumor response by a reduction of tumor size is not applicable and might take months or years to become apparent. Therefore, there is great interest in identifying reliable biomarkers of early tumor response to non-cytotoxic drugs [9]. Imaging techniques could potentially be used as such a biomarker and could provide an early indicator of effectiveness at a functional or molecular level. Concerning morphological imaging of the vasculature, conventional X-ray angiography and digital subtraction angiography (DSA) are widely used in the clinical arena; however, they do not allow for assessment of the microvasculature and thus are not commonly used for response evaluation of antiangiogenic therapies. More promising is the assessment of changes in hemodynamic parameters such as blood flow, blood volume, or vessel permeability. They may be effective biomarkers for response evaluation, because antiangiogenic therapies are designed to affect the abnormal blood vessels found in tumors. Current clinical trials employ various imaging techniques for this purpose, mostly dynamic contrast-enhanced MRI (DCE MRI), and less often ultrasound, PET (especially with [15O] water), and dynamic contrast-enhanced CT (DCE CT) [10]. In the future, targeting specific molecular markers of angiogenesis might also be used for response assessment of antiangiogenic therapies like the VEGF pathway or cell surface markers like the integrin αvβ3.

The role of endothelial cell apoptosis in the effect of etanercept in psoriasis

Psoriasis is a chronic inflammatory skin disease associated with abnormal vascular expansion in the papillary dermis. Tumour necrosis factor (TNF)-α is a proinflammatory cytokine that can induce antiapoptotic proteins and endothelial cell activation factors in psoriasis. The present study investigated the effect of the anti-TNF-α agent etanercept on the expression of endothelial nuclear factor-κB (NF-κB), angiogenic vascular endothelial growth factor (VEGF), endothelial cell marker CD31, antiangiogenic factor thrombospondin-1 (TSP-1), and antiapoptotic factors Bcl-2 and Bcl-xL in psoriasis. Sixteen patients with moderate-to-severe psoriasis were included in the study and treated with etanercept 50 mg twice weekly subcutaneously for 12 weeks. Biopsies of lesional skin (baseline, weeks 3, 6 and 10) were obtained and immunohistochemically stained with antibodies for CD31, VEGF, TSP-1, NF-κB, Bcl-2 and Bcl-xL. Double immunofluorescence staining for VEGF and CD31 was evaluated with confocal laser microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay was applied for apoptosis detection. Etanercept caused a statistically significant time-dependent reduction in the number of dermal blood vessels, the number of CD31+ cells and VEGF in psoriatic lesions, with induction of endothelial cell apoptosis and statistically significant upregulation of TSP-1 in psoriatic vessels. Immunohistochemical analysis showed significant reduction of NF-κB, Bcl-2 and Bcl-xL expression in endothelial cells during treatment. These changes were accompanied by a marked clinical response. The present findings suggest that treatment with etanercept induces apoptosis, reduces apoptosis-inhibiting factors in psoriatic endothelial cells, and decreases angiogenesis in psoriatic skin.

Cost-effectiveness of ranibizumab for the treatment of neovascular age-related macular degeneration in germany: Model analysis from the perspective of germany's statutory health insurance system

Background: In clinical trials, ranibizumab has been associated with stabilization and even improvement of visual acuity among patients with neovascular agerelated macular degeneration (AMD), but its use is also associated with considerable costs. Objective: The aim of this work was to compare ranibizumab with best supportive care or photodynamic therapy (PDT) for AMD by means of economic costutility and cost-effectiveness analysis from the perspective of Germany's Statutory Health Insurance System. Methods: Visual acuity data from the Anti-VEGF (vascular endothelial growth factor) Antibody for the Treatment of Predominantly Classic Choroidal Neovascularization in AMD (ANCHOR) and Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA) studies were applied, based on a ranibizumab dose of 0.5 mg. A Markov model simulated visual acuity and costs over 10 years (discounted at 3%). The base-case analysis assumed 5 injections per year over 2 years. Treatment costs were based on year-2008 euros (using German prices) and recommendations for procedure reimbursement from a public health insurance perspective. To assess cost-effectiveness, costs per year of legal blindness avoided (ie, vision-year gained [VYG]) and per quality-adjusted life-year (QALY) were calculated. The model assumed each patient's affected eye had better sight than the other eye, and the 2 comparators against which ranibizumab treatment was assessed were best supportive care and PDT. The robustness of the results was investigated in a univariate sensitivity analysis of all relevant parameters and a multivariate probabilistic sensitivity analysis. The multivariate 95% CIs for incremental cost-effectiveness ratios were obtained by conducting 1000 Monte Carlo simulations. Results: Compared with best supportive care, costs per VYG for ranibizumab were €6767 in occult choroidal neovascularization (CNV) and €6020 in minimally classic CNV. In classic CNV, costs were €5734/VYG for ranibizumab compared with supportive care and €778/VYG for ranibizumab compared with PDT. Costs per QALY for ranibizumab treatment for occult, minimally classic CNV, and classic CNV were €22,320, €22,538, and €25,036, respectively, and €3294 for classic CNV compared with PDT. Results were sensitive to the cost of blindness, injection frequency, and duration. The multivariate 95% CIs for the incremental costeffectiveness ratios were €14,438 to €41,110/QALY for occult CNV, €13,463 to €43,614/QALY for minimally classic CNV, and €15,634 to €51,106/QALY for classic CNV. Conclusion: In this model analysis using costs and clinical trial data from Germany, ranibizumab appeared to be a cost-effective treatment option for all angiographic subtypes of neovascular AMD, from the perspective of Germany's Statutory Health Insurance System.

Regulation of the blood–brain barrier integrity by pericytes via matrix metalloproteinases mediated activation of vascular endothelial growth factor in vitro

The blood–brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes, and neurons. In this study, we analyzed the influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells in a species-consistent in vitro coculture model. We were able to show a barrier integrity-decreasing impact of pericytes by transendothelial electrical resistance (TEER) and 14C-sucrose permeability measurements. The morphology analysis revealed serrated cell borders and a shift of the endothelial morphology towards a cobblestone shape under the influence of pericytes. The analysis of the two major barrier integrity modulators vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) displayed higher MMP activity and higher levels VEGF, MMP-2, and MMP-9 in the coculture, whereas VEGF levels were decreased by the MMP inhibitor GM6001, indicating a complex interplay of both. Inhibition experiments with neutralizing VEGF antibody and GM6001 increased the TEER, which proves the involvement of VEGF and MMPs in the barrier-decreasing process. Analysis of occludin yielded decreased protein content and discontinuous expression at the endothelial cell borders under the influence of pericytes. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity via MMPs and VEGF.

Angiotensin II upregulates the expression of placental growth factor in human vascular endothelial cells and smooth muscle cells

BackgroundAtherosclerosis is now recognized as a chronic inflammatory disease. Angiotensin II (Ang II) is a critical factor in inflammatory responses, which promotes the pathogenesis of atherosclerosis. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family cytokines and is associated with inflammatory progress of atherosclerosis. However, the potential link between PlGF and Ang II has not been investigated. In the current study, whether Ang II could regulate PlGF expression, and the effect of PlGF on cell proliferation, was investigated in human vascular endothelial cells (VECs) and smooth muscle cells (VSMCs).ResultsIn growth-arrested human VECs and VSMCs, Ang II induced PlGF mRNA expression after 4 hour treatment, and peaked at 24 hours. 10-6 mol/L Ang II increased PlGF protein production after 8 hour treatment, and peaked at 24 hours. Stimulation with Ang II also induced mRNA expression of VEGF receptor-1 and -2(VEGFR-1 and -2) in these cells. The Ang II type I receptor (AT1R) antagonist blocked Ang II-induced PlGF gene expression and protein production. Several intracellular signals elicited by Ang II were involved in PlGF synthesis, including activation of protein kinase C, extracellular signal-regulated kinase 1/2 (ERK1/2) and PI3-kinase. A neutralizing antibody against PlGF partially inhibited the Ang II-induced proliferation of VECs and VSMCs. However, this antibody showed little effect on the basal proliferation in these cells, whereas blocking antibody of VEGF could suppress both basal and Ang II-induced proliferation in VECs and VSMCs.ConclusionOur results showed for the first time that Ang II could induce the gene expression and protein production of PlGF in VECs and VSMCs, which might play an important role in the pathogenesis of vascular inflammation and atherosclerosis.

Mechanisms of enhanced antileukemia activity of conditionally replicating adenovirus (CRAd) ZD55 by interleukin-24

To investigate the mechanisms of enhanced antileukemia activity of conditionally replicating adenovirus (CRAd) by interleukin-24 (IL-24). The ability of CRAd ZD55 to infect leukemia cells was detected by flow cytometry. The expressions of vascular endothelial growth factor (VEGF) in leukemia cells treated with PBS, ZD55, ZD55-IL-24, and an adenovirus carrying IL-24 (Ad-IL-24) were determined by Western blot analysis. Animal xenograft tumor model was established by Mutz-1 cell line.Deparaffinized tumor sections were incubated with anti-CD31, and VEGF antibody, followed by immunohistochemistry analysis. The GFP-positive cells were 5.1% and 42.3% in Mutz-1 cells treated with ZD55-EGFP vector at MOI of 10 and 100 for 48h, respectively. ZD55-IL-24 treatment resulted in the marked down-regulation of VEGF protein expression and ZD55 inhibited VEGF slightly; however, there was no change observed in the cells treated with Ad-IL-24. Immunohistochemistry analysis showed that Ad-IL-24 inhibited slightly angiogenesis and ZD55 treatment resulted in significant inhibition of angiogenesis. ZD55-IL-24 treatment almost completely inhibited angiogenesis in tumor tissues. IL-24 enhances the antileukemia activity of ZD55 by inhibiting VEGF protein expression and angiogenesis in vitro and in vivo.

Intravitreal bevacizumab (Avastin) vs. ranibizumab (Lucentis) for the treatment of age-related macular degeneration: a systematic review

We conducted a systematic review to evaluate whether the existing evidence justifies the intravitreal use of bevacizumab in comparison to ranibizumab in age-related macular degeneration. Compared with photodynamic therapy, bevacizumab shows a relative improvement in visual acuity that is of similar size as in the comparison of ranibizumab with photodynamic therapy (relative improvement from 30 to 35%). However, this finding is based on one randomized controlled trial including less than 50 patients treated with bevacizumab. Also, nothing is known about long-term (>12 months) improvements in visual acuity and optimal treatment intervals for bevacizumab.Regarding safety, the published literature indicates that ocular and systemic adverse effects are less frequent under bevacizumab than ranibizumab treatment. But the validity of this finding is strongly limited by inadequate reporting, an unsystematic evaluation of adverse effects and short follow-up times in studies evaluating bevacizumab. Given the lack of controlled data, the widespread off-label use of bevacizumab is not justified in clinical practice. On the other hand, a major challenge in the management of patients who require repeated antivascular endothelial growth factor injections is the high cost of ranibizumab. This dilemma underlines the need for head-to-head studies comparing both vascular endothelial growth factor antibodies, or, at least, well conducted randomized controlled trials evaluating intravitreal bevacizumab.

Abstract 5304: Serum vascular endothelial growth factor in advanced breast cancer patients might be a key role of inhibitory effect on lymphokine activated killer cells

Abstract Previously, we reported that serum from stage IV breast cancer patients diminished the cytotoxic activity of lymphokine activated killer cells (LAKs) (Abstract #4639 AACR in 2008). Thus, we next tested what the critical role for the inhibitory effect on LAKs was. Based on the study of protein detection in the serum using Cytokine Antibody Array (Ray Biotech), serum from stage IV patients seemed to have higher level of vascular endothelial growth factor (VEGF) as compared to early stage patients. To confirm this by more accurate way, we analyzed the serum level of VEGF by using ELISA in 24 breast cancer patients. When the serum level of VEGF was cut off by median (420 pg/ml), 11 patients with higher level of VEGF out of 12 are included in the patients whose serum inhibited the cyototoxic activity of LAKs. Therefore, we hypothesized that VEGF might be one of the key role for inhibitory effect on LAKs and neutralization of serum VEGF from the patients whose serum inhibited the cyototoxic activity of LAKs could have no inhibitory effect on LAKs. Briefly, PBMCs from a healthy volunteer were used as an effector cell for all patients’ serum incubation. After activation of the PBMCs using IL-2, LAKs were prepared and incubated with the individual patient's serum and washed. The LAKs were incubated with human breast cancer cell line MCF-7 as a target cell and LDH assay was performed to detect cytotoxicity of the LAKs. Fourteen out of 24 patients serum (serum 1) showed inhibitory effect on LAKs and we picked up these patients to test blocking study of serum VEGF. Briefly, serum from these 14 patients was incubated with neutralization antibody of VEGF (R16Y, Santa Cruz Biotechnology) (serum 2) before mix with LAKs. After incubation of serum 1 and serum 2 with LAKs, target cell cytotoxicity of LAKs from incubation with serum 1 showed 30-50% reduced level as compared to control LAKs activity. Interestingly, target cell cytotoxicity of all LAKs from incubation with serum 2 did not show such inhibitory effect (mean cell cytotoxicity of LAKs was 33.48% and 57.27%, serum 1 and serum 2, respectively, control; 55.73%). These data suggest that high level of serum VEGF in breast cancer patients might be a key role of inhibitory effect on LAKs and neutralization of the VEGF could help adoptively transferred LAKs as cancer immunotherapy in the patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5304.

A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.

Phage display peptide probes for imaging early response to bevacizumab treatment

Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized vascular endothelial growth factor antibody bevacizumab. Using a bevacizumab-sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage) display peptide library, a bevacizumab-responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo. This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab-treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800- and (18)F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate-buffered saline. Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature. In summary, a novel 12-mer peptide BRP selected using phage display techniques allowed non-invasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.

Role of myeloid cells in vascular endothelial growth factor-independent tumor angiogenesis

Targeting the vascular endothelial growth factor (VEGF) pathway has had a significant impact in the therapy of cancer and intraocular neovascular disorders. Similar to other therapies, inherent/acquired resistance to anti-VEGF drugs may occur in cancer patients, leading to disease recurrence. This review describes recent findings on the role of myeloid cells in refractoriness or/and acquired resistance to such therapies. Various myeloid cell types, including tumor-associated macrophages, Tie2-expressing monocytes and neutrophils, have been implicated in tumor angiogenesis. Several cytokines involved in the mobilization and/or proangiogenic effects of these cells represent therapeutic targets. CD11b+Gr1+ myeloid cells have been shown to render tumors refractory to angiogenic blockade by VEGF antibodies. This effect was mediated by the secreted protein Bv8, which is upregulated by granulocyte colony-stimulating factor. Progress in unraveling proangiogenic mechanisms dependent on various myeloid cell types has expanded our understanding of tumor angiogenesis and has generated a number of potential therapeutic targets.

Vascular Endothelial Growth Factor and Its High-Affinity Receptor (VEGFR-2) Are Highly Expressed in the Human Forebrain and Cerebellum During Development

Vascular endothelial growth factor (VEGF) is an angiogenic and neurotrophic factor in both adult and neonatal animals, but its expression and role have been incompletely studied in the developing human brain. We analyzed the distribution of VEGF and its high-affinity receptor VEGFR-2 in the human forebrain and cerebellum at developmental stages from 14 weeks' gestation (WG) to the13th postnatal month. Tissue samples free of detectable neuropathologic abnormalities were assessed by immunohistochemistry and confocal microscopy using anti-human VEGF and VEGFR-2 antibodies. The VEGFR-2 was first expressed in the whole cerebral mantle and in migrating cells in the intermediate zone, whereas VEGFwas found in superficial layers of the cortical plate, in radial glia, and in the cerebellar external germinal cell layer. From 23 WG, temporospatial VEGFR-2 expression was superimposable on that ofVEGF in the cortical plate, intermediate zone, basal ganglia, limbicstructures, and external germinal cell layer. The VEGF/VEGFR-2-positive astrocytes were observed during their generation and migration from 23 WG to the first postnatal month. The VEGF-positive mature oligodendrocytes were observed in myelinating structures in the forebrain from birth and in the cerebellum from 24WG. These data suggest that VEGF and VEGFR-2 are likely involved in several aspects of human brain development.