anti-KDR Antibody Research Articles

Construction and measuring combination of KDR-targeted ultrasound contrast agent in vitro for evaluating endometrial receptivity

Objective: To investigate the preparation of a new kind of targeted lipid ultrasound contrast agent with anti-KDR antibody based on biotin- avidin bridge (MB-BAB-KDR) which could combine specifically with KDR that increases during the time of embryo implantation. Then its binding capability in vitro was evaluated. Materials and Methods: The agitation of high-speed method was employed to prepare biotin-microbubbles (MB-B), and biotin–avidin mediated technique was used to produce MB-BAB-KDR. MB-BAB-KDR, MB-B, and biotin- microbubbles-KDR (MB-B-KDR) were incubated with fluorescein-conjugated affiniPure goat anti-rat IgG (H+L) to assess the linked condition. Second, MB-BAB-KDR and control groups (MB-B and MB-B-KDR) were incubated with human umbilical vein endothelial cell (HUVEC). Rosette formation rate was observed and calculated. Then, the parallel plate flow chamber technology was used to access attachment efficiency to KDR Fc. Results: The surface of bubbles could carry KDR antibody through “biotin-avidin” bridge. After incubated with second antibody, bright green fluorescence (II grade) could be observed in MB-BAB-KDR group, as compared with weak fluorescence in control groups of MB-B (0 grade) and MB-B-KDR (I grade). The surrounding rosette formation rate on HUVEC was 89.86% in MB-BAB-KDR group and that of control groups were 7.13% (MB-B-KDR) and 3.02% (MB-B) (p p Conclusion: The successful preparation of MB-BAB-KDR with anti-KDR antibody which shows specially targeting binding capability with HUVEC and stability in shear stress may be served as a noninvasive detection of endometrial vascular KDR expression and provide an experimental foundation for evaluating endometrial receptivity in vivo.

Decreased efficacy of drugs targeting the vascular endothelial growth factor pathway by the epigenetic silencing of FLT1 in renal cancer cells

BackgroundThe vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) signaling pathway is involved in cancer-related biological functions and is a therapeutic target in cancer. However, the influence of epigenetic regulation of VEGF-VEGFR signaling-related genes remains unclear. Here, we evaluated the effects of FLT1 and KDR promoter hypermethylation combined with drugs targeting VEGF-VEGFR signaling on cancer-related phenotypes in renal cancer cells (RCCs) and examined changes in FLT1 and KDR promoter hypermethylation in tissues from patients with renal cancer.ResultsIn vitro experiments were performed to evaluate the effects of beavacizumab (an anti-VEGF antibody), an anti-FLT1 peptide, an anti-KDR antibody, and the VEGFR tyrosine kinase inhibitors (TKIs) sunitinib and axitinib in 13 RCC lines with different levels of FLT1 and/or KDR promoter methylation and in 2 FLT1 or KDR in vitro knockdown models. The synergistic effects of sunitinib and axitinib treatment were also evaluated in four RCC lines having different levels of FLT1 and/or KDR methylation. In our in vitro experiments, bevacizumab and an anti-KDR antibody did not affect the proliferation of RCCs having FLT1 and/or KDR hypermethylation. In contrast, in RCCs with FLT1 hypermethylation, proliferation inhibition was counteracted by treatment with an anti-FLT1 peptide and both VEGF-TKIs (sunitinib and axitinib). Demethylation with sunitinib or axitinib synergistically increased proliferation inhibition in the RCCs exhibiting FLT1 hypermethylation. Using in vitro FLT1 or KDR knockdown models, decreased proliferation inhibition following anti-FLT1 peptide, sunitinib, and axitinib treatment was observed only in FLT1-knockdown cells. In patients with renal cancer who received sunitinib, FLT1 promoter methylation was higher in renal cancer tissues from eight nonresponders (stable or progressive disease assessed by the Response Evaluation Criteria in Solid Tumors) than in cancer tissues from five responders (complete response or partial response).ConclusionsThe present data showed that hypermethylated FLT1 was important for the efficacy of anti-VEGF/VEGFR drugs targeting FLT1 or intracellular VEGFR signaling. FLT1 hypermethylation causing alterations of FLT1 function could serve as a useful biomarker for predicting changes in FLT1 status in RCCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0134-9) contains supplementary material, which is available to authorized users.

The patterns and expression of KDR in normal tissues of human internal organs

KDR has been implicated for playing an important role in the formation of new blood vessels and in solid tumor growth. It was considered as one of the most important regulators of angiogenesis and a key target in anticancer treatment. In the present study, we characterized KDR mRNA and protein expression in normal tissues of perinatal and adult tissues using One-step Real-Time RT-PCR and immunohistochemistry with a self-made anti-KDR antibody. The expression of KDR mRNA and protein in perinatal internal organs were all higher than in adult organs including brain, kidney, liver, lung and heart, respectively. KDR protein was presented in the cell plasma membrane of human internal tissues. The expression of KDR protein was raised in macrophage of spleen, and decreased in neurons of brain, myocardium, bronchial epithelial cells and alveolar epithelial cell, proximal and distal tubules cells, and hepatic cells with the maturity process of human organs. Notably, the order of KDR protein expression from highest to lowest is as follows: brain, liver, heart, kidney, and lung in adult tissues with statistically significant. It follows that how to balance the potential therapeutic side effect with human internal organs in targeted therapy of over-expressing KDR tumor.

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

Comparison of Endothelial Progenitor Cells in Parkinson's Disease Patients Treated with Levodopa and Levodopa/COMT Inhibitor

BackgroundLevodopa treatment in Parkinson's disease (PD) increases in serum homocysteine levels due to its metabolism via catechol O-methyltransferase. Endothelial progenitor cells (EPCs) have the capacity to differentiate into mature endothelial cells and are markers for endothelial functions and cardiovascular risks. Along with traditional vascular risk factors, hyperhomocysteinemia is known to decrease the level of EPCs. In the present study, we hypothesized that that levodopa-induced hyperhomocysteinemia leads to a change in EPC levels.Methodology/Principal FindingsWe prospectively enrolled PD patients who had been prescribed either levodopa/carbidopa (PD-L group, n = 28) or levodopa/carbidopa/COMT inhibitor (PD-LC group, n = 25) for more than 1 year. The number of circulating EPCs was measured by flow cytometry using dual staining of anti-CD34 and anti-KDR antibodies. The EPCs were divided into tertiles based on their distributions and a logistic regression analysis was used to estimate independent predictors of the highest tertile of EPCs. The number of endothelial progenitor cells was significantly decreased in PD-L patients (118±99/mL) compared with either PD-LC patients (269±258/mL, p = 0.007) or controls (206±204/mL, p = 0.012). The level of homocysteine was significantly increased in PD-L patients (14.9±5.3 µmol/L) compared with either PD-LC patients (11.9±3.0 µmol/L, p = 0.028) or controls (11.1±2.5 µmol/L, p = 0.012). The level of homocysteine was negatively correlated with endothelial progenitor cell levels (r = −0.252, p = 0.028) and was an independent predictor of the highest tertile of endothelial progenitor cell levels (OR; 0.749 [95% CI: 0.584–0.961]).Conclusions/SignificanceThese data indicate that a higher consumption of EPC for restoration of endothelial damage may be associated with chronic levodopa treatment in PD patients.

Abstract 4252: The addition of bevacizumab to paclitaxel overcomes chemoresistance to paclitaxel in a class III beta tubulin expressing gastric cancer cells

Abstract Background: Class III β tubulin (TUBB3) reduces microtubule stability and confers resistance to microtubule stabilizing taxanes, including paclitaxel and docetaxel. Expression of vascular endothelial growth factor (VEGF) is highly regulated by hypoxia and the regulation of VEGF expression by hypoxia is mediated by a hypoxia inducible factor-1a(HIF-1a), which increase transcription of the VEGF gene. Recent report showed hypoxia induces expression of TUBB3 by HIF-1a. Inhibition of VEGF system may decrease the expression of HIF-1a and TUBB3. This study was undertaken to investigate the synergistic antitumor effects of bevacizumab and palitaxel on TUBB3 expressing gastric cancer cells. Methods: We evaluated the expressions of TUBB3 and HIF-1a under normoxic and hypoxic conditions in human gastric cancer cells. The chemoresistance of TUBB3 to paclitaxel was checked through knockdown of TUBB3. Expressions of TUBB3 and HIF-1a were measured under blocking of VEGFR1/2 with anti-FLT1 and anti-KDR antibodies. The effects of bevacizumab and paclitaxel on expressions of TUBB3 and HIF1a were monitored by western blot and MTT assay. Results: The chemosensitivities to paclitaxel were increased by siTUBB3. Elevated expressions of TUBB3 and HIF-1a in gastric cancer cells under hypoxic conditions were reduced in the prescence of anti-KDR and anti-FLT1 antibodies. When we added the bevacizumab to paclitaxel, the protein expressions of TUBB3 and HIF-1a in gastric cancer cells under hypoxic conditions were significantly decreased as compared with paclitaxel alone. Apoptosis of gastric cancer cells were more increased in a combined treatment with bevacizumab and paclitaxel than paclitaxel alone. Tyrosine phosphorylation of Erk and Akt was also measured in gastric cancer cells under hypoxic conditions in the absence or prescence of anti-KDR, anti FLT-1 and paclitaxels. Hypoxia induced phosphorylation of Erk and Akt in gastric cancer cells was more decreased by addition of anti FLT-1 and anti KDR antibodies to paclitaxel as compared with paclitaxel alone. Conclusion: Our experiments show that a VEGF/VEGFR/pERK and, or pAKT pathway is activated by hypoxic gastric cancer cells to induce HIF-1a and TUBB3 expressions. Combined treatment with paclitaxel and bevacizumab, a recombinant humanized monoclonal antibody that binds to VEGF, synergistically decreased the expressions of HIF-1a and TUBB3 in gastric cancer cells in hypoxic conditions. With increasing use of taxanes in the treatment of gastric cancer, this study showed the potential synergistic antitumor effects of bevacizumab and paclitaxel in a TUBB3, a marker of resistance to paclitaxel, overexpressing gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2011-4252

Abstract 4244: TTAC-0001, fully human anti-VEGFR2/KDR neutralizing antibody blocks tumor angiogenesis and tumor growth

Abstract Angiogenesis, the recruitment of new blood vessels is a crucial mechanism required for both tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptors, particularly VEGF receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in tumor-associated angiogenesis. Monoclonal antibodies (mAb), due to their high specificity towards a given target, represent a unique class of novel therapeutics as angiogenesis inhibitors. We developed TTAC-0001, anti-KDR antibody from a fully human naïve single chain variable fragment (ScFv) phage library. Interestingly, TTAC-0001 displayed cross reactivity against murine homologue, Flk-1, which allows us to evaluate the anti-tumor efficacy in various in vivo models. TTAC-0001 inhibits VEGF-mediated proliferation and migration. In HUVEC, TTAC-0001 inhibited the phosphorylation of VEGFR2/KDR and ERK mediated by VEGF. TTAC-0001 inhibited VEGF-mediated sprouting endothelial cells from rat aortic rings and showed a potent anti-angiogenic activity in VEGF-mediated matrigel model in nude mice. Further in vivo anti-tumor efficacy of TTAC-0001 was seen in the xenografts of A549 (lung) and HCT116 (colorectal) human cancer. These changes were accompanied by the inhibition of microvessel density and induction of apoptosis in the tumors. In summary, our data indicates that TTAC-0001 blocks the binding of VEGF to VEGFR2/KDR and inhibits VEGFR-induced angiogenesis. Therefore, this data strongly supports for the further development of TTAC-0001 as an anti-cancer agent (This study was supported by grants of the Korea Healthcare technology R&D Project, Ministry for Health & Welfare Affairs (A092255 and A040016) and Advanced Medi-cluster R&D Project, Daejeon TechnoPark, Republic of Korea). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2011-4244

Effect of vascular endothelial growth factor and its receptor KDR on the transendothelial migration and local trafficking of human T cells in vitro and in vivo

AbstractIn these studies, we find that the vascular endothelial growth factor (VEGF) receptor KDR is expressed on subsets of mitogen-activated CD4+ and CD8+ T cells in vitro. We also found that KDR colocalizes with CD3 on mitogen-activated T cells in vitro and on infiltrates within rejecting human allografts in vivo. To evaluate whether VEGF and KDR mediate lymphocyte migration across endothelial cells (ECs), we used an in vitro live-time transmigration model and observed that both anti-VEGF and anti-KDR antibodies inhibit the transmigration of both CD4+ and CD8+ T cells across tumor necrosis factorα (TNFα)–activated, but not unactivated ECs. In addition, we found that interactions among CD4+ or CD8+ T cells and TNFα–activated ECs result in the induction of KDR on each T cell subset, and that KDR-expressing lymphocytes preferentially transmigrate across TNFα–activated ECs. Finally, using a humanized severe combined immunodeficient mouse model of lymphocyte trafficking, we found that KDR-expressing lymphocytes migrate into human skin in vivo, and that migration is reduced in mice treated with a blocking anti-VEGF antibody. These observations demonstrate that induced expression of KDR on subsets of T cells, and locally expressed VEGF, facilitate EC-dependent lymphocyte chemotaxis, and thus, the localization of T cells at sites of inflammation.

Role of bevacizumab on class III beta tubulin and HIF-1a in paclitaxel-treated gastric cancer.

e13656 Background: At present, there are eight beta tubulin isotypes in mammalian cells. Among these isotypes, class III beta tubulin was shown to confer resistance to antitubulin agents, such as paclitaxel or docetaxel. Of the inducer of class III beta tubulin (TUBB3), recent report showed that HIF-1a (hypoxia inducible factor-1alpha), one of contributor to chemoresistance in gastric cancer. And HIF-1a was suppressed in addition with anti-vascular endothelial growth factor, bevacizumab, in hypoxia sensitive cell line. This study was undertaken to investigate the role of bevacizumab on expression of class III beta tubulin and HIF-1a. Methods: We evaluated TUBB3 and HIF-1a expression under normoxic and hypoxic condition in human gastric cancer cell lines. The role of TUBB3 to antitumor effect of paclitaxel was checked through knockdown of TUBB3. TUBB3 and HIF-1a were measured under blocking of VEGFR1/2 with anti-FLT1 and anti-KDR antibody. The effects of bevacizumab and paclitaxel on TUBB3 and HIF1a expression were monitored by western blot and MTT assay. Results: The chemoresistance of paclitaxel was overcome by siTUBB3. In hypoxic condition, elevated expression of TUBB3 and HIF-1a were reduced by treating with anti-KDR and anti-FLT1 antibody. Compare to paclitaxel alone, when added bevacizumab to paclitaxel, the protein expressions of TUBB3 and HIF-1a were significantly decreased. Also, cell proliferation was more reduced in bevacizumab and paclitaxel combination than paclitaxel alone. Conclusions: Previously, VEGF has been well known as a target molecule of HIF-1a downstream signals. However, we showed that VEGF can also control HIF-1a as an upstream signal. Bevacizumab, which is blocker of VEGF, and paclitaxel synergistically suppressed HIF-1a and TUBB3. This study showed the potential key role of combination chemotherapy with bevacizumab and paclitaxel in TUBB3 expressed cancer. No significant financial relationships to disclose.

Preparation of extracellular domain 3 of human VEGF receptor‐2 and the monitoring of its real‐time binding to VEGF by biosensors

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessels, which is involved in tumor angiogenesis, a process that is essential for tumor growth and metastasis. In this study, we describe a method for high yield of recombinant extracellular domain 3 (KDR3) of human VEGFR-2 in an Escherichia coli system with further purification by cation exchange chromatography and immobilized metal affinity chromatography (IMAC). The biological activity of recombinant KDR3 was performed by sequestering VEGF in HUVEC proliferation assay. The real-time binding of human VEGF to immobilized KDR3 was monitored by a label-free biosensor, Optical waveguide lightmode spectroscopy (OWLS). Under the given experimental conditions, the association rate constant k(a) was 4.2 x 10(3) M(-1) s(-1) and the dissociation rate k(d) was 5.1 x 10(-3) s(-1). The dissociation constant K(D) was then calculated to be 1.2 x 10(-6) M. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptors and the evaluation of developed anti-KDR antibodies. In addition, such a scenario established by the use of OWLS will potentiate the kinetic study of ligand/receptor and antigen/antibody. The receptor discussed here, which block VEGF binding to cell membrane KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.

Effect of anti-KDR antibody on the proliferation of hemangioma vascular endothelial cells in vitro

The suppressive effect of anti-KDR antibody against VEGF on proliferation of hemangioma-derived vascular endothelial cells (HVECs) was investigated. HVECs from one case of hemangioma in proliferative phase were cultured. Both primary culture and sub-culture were conducted in M199 medium. The HVECs of passage 3 were divided into 4 groups based on the concentrations of anti-KDR antibody. Cell count was performed and inhibitory rate of HVECs was measured before and 9 days after interference. The results showed that the number of HVECs in the anti-KDR antibody-treated groups was significantly decreased and the inhibitory rate of HVECs by anti-KDR antibody (50, 10 and 2 microg/mL) was 84%, 63% and 39% respectively at 9th day after interference, with the difference being significant. In the control group, the number of HVECs was increased significantly. In was concluded that the anti-KDR antibody could suppress the activity of VEGF through blocking the KDR, indicating the potential clinical applications of anti-KDR antibody in the treatment of hemangioma.

Engineering of heart valves using circulating progenitor cells from patients with valvular disease ‐ A feasibility study

The potential of using progenitor-derived endothelial (ECs) and muscle (SMCs) cells in the tissue-engineering of cardiac valve has been explored in the present study. Circulating progenitor cells were isolated from blood samples of patients with valvular disease. Cells were identified using anti-CD133 and anti-KDR antibodies, expanded and differentiated into ECs and SMCs that were characterized immunohistochemically. ECs stained positive for CD31, VE-cadherin and vWF, and formed capillaries in matrigelTM. SMCs stained positive for α-actin and caldesmon. Collagen matrices prepared form decellularized porcine cardiac valves were used to develop an engineered valve. When seeded on to these matrices, ECs and SMCs attached, proliferated and formed defined cell layers. The engineered valve showed similar biomechanical properties as normal valve tissues. The degree of coagulation was minimal in cell seeded valve surfaces, when compared to unseeded matrices. This study shows the feasibility of isolating and culturing progenitor cells from patients that could be differentiated into ECs and SMCs. Progenitor-derived cells retain their characteristcs and could be used for engineering of cardiac valves for autologous implantation.

Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naïve human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

Inhibition of Both the Autocrine and the Paracrine Growth of Human Leukemia with a Fully Human Antibody Directed Against Vascular Endothelial Growth Factor Receptor 2

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Here we show that certain "liquid" tumors such as acute myeloid leukemia not only produce VEGF but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. In addition, the leukemia-derived VEGF can also stimulate the production of growth factors, including interleukin 6 (IL6) and granulocyte-macrophage colony stimulating factor (GM-CSF), by human endothelial cells, which in turn further promotes the growth of leukemia cells (the paracrine loop). A fully human anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-2C6, strongly blocks KDR/VEGF interaction and neutralizes VEGF-stimulated activation of KDR in endothelial cells. In a system where leukemia cells are co-cultured with endothelial cells, IMC-2C6 inhibits both the production of IL6 and GM-CSF by endothelial cells and the growth of leukemia cells. Finally, IMC-2C6 effectively blocks VEGF-induced migration of KDR+ human leukemia cells, and when administered in vivo, significantly prolonged survival of mice inoculated with KDR+ human leukemia cells. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and certain types of leukemia.

Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell mitogen involved in normal and pathological angiogenesis. Our group recently reported that, among the several VEGF isoforms, VEGF189 (V189) is selectively induced in decidual endometrial cells during the mid-late phase of the menstrual cycle, together with polymorphonuclear neutrophil (PMN) influx. We thus compared the effects of various VEGF isoforms on PMN migration in vitro, and the mechanisms involved. In transmigration and under-agarose assays, V189 was both chemotactic and chemokinetic for PMN, while VEGF165 (V165) was only chemokinetic. The chemokinetic effect of V189 for PMN was blocked by neutralizing anti-VEGF antibodies, but not by neutralizing anti-KDR antibodies, suggesting that the Flt-1 VEGF receptor that is expressed in PMN mediates these effects. Flow cytometric analysis of several adhesion molecules at the PMN surface showed that all VEGF isoforms slightly upregulated β1- and β2-integrins and PECAM, and downregulated L-selectin; all these molecules are activation markers. The involvement of β1-integrins was further supported by the ability of blocking antibodies to reduce VEGF-induced PMN migration. As human PMN can secrete several cytokines and growth factors, the selective secretion of VEGF isoforms was also further examined. RT-PCR analysis showed that V165 mRNA was more strongly expressed than V189 mRNA. Conversely, the major protein isoform secreted after optimal PMN degranulation was V189, which was located in both azurophilic and specific granules. PMN-derived VEGF can thus modulate PMN migration. This autocrine amplification mechanism would allow sustained VEGF release to occur at inflammatory sites, and may contribute to both normal and pathological angiogenesis.

Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus.

Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor in establishing the vasculature in renal cell carcinomas (RCCs). Since little is known about VEGF signalling in RCCs, the profile of phosphorylated KDR (pKDR) has been investigated and the intracellular location of the receptor has been examined in the present study. Using two monoclonal antibodies raised against the phosphorylated KDR epitopes (Y1059 and Y1214) known to mediate different VEGF functions, together with a commercial anti-KDR antibody and immunohistochemistry, the expression of pKDR was investigated in a series of normal (n = 25) and neoplastic kidneys (n = 54; clear cell n = 35; papillary n = 10; oncocytomas n = 8). pKDR was present in many tissue elements of both normal and neoplastic renal tissues, with strong expression in the cell membrane, cytoplasm, and nuclei of normal kidney and tumour cells, as well as endothelial cells in tumours of all histological types. Patterns and intensity were similar using both anti-pKDR antibodies. There was no significant correlation in clear cell carcinomas between pKDR expression and age (p = 0.57), tumour size (p = 0.2), gender (p = 0.59), grade (p = 0.2) or histological type (p = 0.36). To delineate further the intracellular processing that might account for the cellular distribution, confocal microscopy was also performed. Antibodies to the different phosphorylated epitopes demonstrated different intracellular staining patterns. This study shows that pKDR is present in a wide variety of renal tumours, suggesting that anti-VEGF therapy might have direct effects on tumour cells. It further suggests that cells traffic pKDR depending on the precise KDR tyrosines that are autophosphorylated in a manner that enables receptor activation to result in different functions.

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.

NCK and PAK Participate in the Signaling Pathway by Which Vascular Endothelial Growth Factor Stimulates the Assembly of Focal Adhesions

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.

VEGF protects against oxidized LDL toxicity to endothelial cells by an intracellular glutathione-dependent mechanism through the KDR receptor.

Although the accumulation of vascular endothelial growth factor (VEGF) has been observed in human atherosclerotic lesions, the exact role of this growth factor in atherogenesis remains unknown. We hypothesized that VEGF in the vascular wall might have a preventive effect on endothelial cell damage during atherosclerosis. To test our hypothesis, we examined whether VEGF protects against the toxicity of oxidized low density lipoprotein (Ox-LDL) in cultured endothelial cells derived from bovine aortas (BAECs). Preincubation of BAECs with VEGF prevented Ox-LDL-induced toxicity in a preincubation time- and VEGF concentration-dependent manner. Addition of N(omega)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not reverse the protective effect of VEGF on Ox-LDL toxicity. Incubation of BAECs with VEGF increased intracellular glutathione (GSH) content in a time-dependent manner. Combined addition of VEGF and L-buthionine sulfoximine, a GSH synthesis inhibitor, reversed both GSH levels and the protective effect of VEGF on Ox-LDL-induced cytotoxicity. Placenta growth factor, which ligates to the VEGF Flt-1 receptor but not KDR/Flk-1, failed to prevent Ox-LDL toxicity and had no effect on intracellular GSH levels. An anti-KDR antibody completely blocked these beneficial activities of VEGF. These results suggest that VEGF prevents Ox-LDL-induced endothelial cell damage via an intracellular GSH-dependent mechanism through the KDR/Flk-1 receptor.