Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies selected from an immune b9 allotype rabbit antibody library

Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Models of murine tumor angiogenesis and receptor-specific antibodies are required to evaluate roles of VEGF receptors in mouse models of human cancer. Human VEGFR2 (also known as KDR) and murine VEGFR2 (or Flk-1) share 85% amino acid sequence identity in their extracellular domain. We describe here the development of antibodies that cross-react with mouse and human VEGFR2. High-affinity, species cross-reactive, Fabs specific for KDR/Flk-1 were selected from an antibody phage display library generated from an immunized rabbit of b9 allotype. The selected chimeric rabbit/human Fabs were found to bind to purified KDR and Flk-1 with nanomolar affinity. Three of the selected Fabs detected KDR expression on human endothelial cells as well as Flk-1 on murine endothelial cells. The availability of anti-VEGFR2 Fab with species cross-reactivity will help to decipher the functional role of KDR/Flk-1 in tumor biology as well as facilitate the preclinical evaluation of the suitability of KDR/Flk-1 for drug targeting. This report underscores our earlier finding that b9 rabbits are excellent sources for high-affinity cross-reactive antibodies with therapeutic potential.     

Role of signals from the dorsal root ganglion in neuropathic pain in a rat model

Vascular endothelial growth factor (VEGF), known as an endothelial cell-specific mitogen, has been reported to be linked also to the NO/cGMP-pathway, which has been notified in the inner ear. Up to now, VEGF has not yet been described in the inner ear. We performed immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF-receptors Flt-1 and KDR/Flk-1 on paraffin-sections of temporal bones from guinea pigs (n=5). Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in a subpopulation of vestibular ganglion cells. VEGF could be found also in the endothelium of blood vessels, in fibrocytes of the lamina propria and in the neuroepithelium. Strong immuno-labelling to Flt-1 was evident in nerve fibres, vascular endothelium and in the neuroepithelium. Fibrocytes, endothelium of blood vessels, supporting cells and calyces in the sensory epithelium revealed immunoreactivity to KDR/Flk-1. These findings give evidence that VEGF, Flt-1 and KDR/Flk-1 are constitutively expressed in the vestibule.     

Specific transduction of HIV-1 envelope expressing cells by retroviral vectors pseudotyped with hybrid CD4/CXCR4 receptors

Infection of a target cell by HIV is initiated by the interaction of the envelope glycoprotein with the CD4 receptor molecule on the surface of the target cell. This is followed by binding of a coreceptor of the chemokine receptor family and subsequently fusion of viral and cellular membranes. Membrane fusion is independent of whether the viral envelope protein is on the viral or on the cellular membrane. Accordingly, targeting of HIV infected cells by retroviral vectors has been previously achieved both by coincorporation of CD4 and coreceptors into murine leukemia virus (MLV) and lentivirus based vector particles. It was, therefore, tested whether hybrid genes of CD4 and CXCR4 are also able to yield 'receptor' vectors. A construct containing the four extracellular loops of CD4 fused to CXCR4 (CD4-D4-X4) allowed gene transfer into HIV-1 envelope expressing cells by vectors based on either MLV or lentiviruses. The CD4-D2-X4 hybrid receptor, containing the first two extracellular CD4 domains, allowed gene transfer only by lentiviral vectors. Attempts to increase vector titres by deletion of the intracellular part of CXCR4 failed. Vector titres obtained by hybrid receptors were slightly lower than published titres obtained by separate expression of CD4 and CXCR4. Thus, CD4-D4-CXCR4 hybrids are useful for the generation of retroviral and lentiviral vectors with specificity for HIV-1 envelope expressing cells.     

Expression of growth factors,growth inhibiting factors,and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops

The aim of the present study was to investigate which growth factors, receptors, and growth inhibiting factors are expressed in invasive breast cancer. Five (angiogenic) growth factors and their receptors: platelet-derived growth factor A chain (PDGF-AA) and PDGF receptor alpha (PDGFαR), PDGF-BB and PDGF beta receptor, transforming growth factor alpha (TGFα) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor I (Flt-1) and vascular endothelial growth factor receptor II (Flk-1/KDR); two growth inhibiting factors: transforming growth factor beta-1 (TGFβ1) and TGFβ2) and their receptor couple transforming growth factor beta receptor I (TGFβR-I) and TGFβR-II; and basic fibroblast growth factor (bFGF) were stained by standard immunohistochemistry on frozen sections in 45 cases of invasive carcinoma of the breast. Staining was scored as negative or positive in tumour epithelium, stroma, and blood vessels. TGFβ1 and TGFβ2 were expressed in the tumour cells in 67 per cent and 76 per cent of cases, respectively, whereas PDGFβR and TGFβR-II were expressed in 0 per cent and 2 per cent, respectively. The other factors showed variable expression in tumour cells. All factors were expressed in the stroma in most cases, except Flt-1, Flk-1/KDR, TGFβ2, and TGFβR-II, which showed variable expression, and EGFR, which showed no expression. The endothelium was in most cases positive for bFGF, PDGF-AA, PDGF-BB, VEGF, PDGFαR, PDGFβR, and TGFβ1 but TGFβ2 was negative in most cases and TGFα, EGFR, Flt-1, Flk-1/KDR, TGFβR-I, and TGFβR-II were variably expressed. The most interesting possible auto/paracrine loops, as demonstrated on serial sections and by fluorescence double staining, were the TGFα/EGFR, TGFβs/TGFβR, VEGF/Flt-1, and the VEGF/Flk-1 combinations. In conclusion, growth factors, growth inhibiting factors, and their receptors are frequently expressed in invasive breast cancer. Indications for some possible auto-and paracrine loops have been found, which should encourage further study on the role of these factors in breast cancer proliferation and angiogenesis. © 1998 John Wiley & Sons, Ltd.     

胰岛素对牛胸主动脉内皮细胞血管内皮生长因子及其受体表达的影响

目的:评价胰岛素对培养的牛胸主动脉内皮细胞血管内皮生长因子(VEGF)及其受体表达的影响。方法: 取新生的小牛胸主动脉,做血管内皮细胞原代及传代培养,取4-6代培养细胞分组,应用不同浓度的胰岛素(30 mU/L、300 mU/L、3 000 mU/L)干预培养过程,48 h后应用免疫组化法测定内皮细胞VEGF及其受体(flt-1、flk-1/KDR)的表达水平。结果: 低浓度胰岛素组(30 mU/L、300 mU/L)内皮细胞VEGF表达明显高于不用胰岛素组(P<0.01);高浓度组(3 000 mU/L)内皮细胞VEGF表达明显低于不用胰岛素组(P＜0.05);各组内皮细胞VEGF受体(flt-1及flk-1/KDR)的表达无明显差异(P＞0.05)。结论: 低浓度胰岛素促进小牛主动脉血管内皮细胞VEGF表达;高浓度胰岛素可抑制血管内皮细胞VEGF表达;胰岛素对小牛主动脉血管内皮细胞VEGF受体(flt-1、flk-1/KDR)的表达无直接影响。     

Promotion of retroviral entry in the absence of envelope protein by chlorpromazine

Retrovirus packaging cell lines that express the Moloney murine leukemia virus gag, pol, and env genes and a retroviral vector genome can produce virus particles that are capable of transducing cells. Normally if the packaging cell line does not produce a functional viral fusion glycoprotein, such as the retroviral envelope protein or a foreign viral glycoprotein, then the viruses will be incapable of transducing cells. We have found that incubating envelope protein-deficient virus particles bound to cells with chlorpromazine leads to transduction. Chlorpromazine (CPZ) is a membrane-active reagent that is commonly used to induce the hemifusion to fusion transition when membrane fusion is mediated by partially defective viral glycoproteins. The concentration and pH dependence of the promotion of transduction by CPZ is consistent with a role for CPZ micelle formation in viral entry. These data indicate that caution is warranted when experiments concerning membrane fusion completion promoted by CPZ are analyzed.     

Tumor specific activation of the VEGF/KDR angiogenic pathway in a subset of locally advanced squamous cell head and neck carcinomas

Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 and flk-1(KDR), constitute an important angiogenic pathway which, under hypoxic conditions, is up-regulated in many solid tumours. We used the monoclonal antibody 11B5, specific for recognizing VEGF expression and the `VEGF/flk-1(KDR) complex' on tumour endothelium, to assess free VEGF protein expression and VEGF/receptor activated microvessel density (aMVD) in a series of 104 inoperable locally advanced squamous cell carcinomas of the head and neck, treated with chemo-radiotherapy. High VEGF expression in cancer cells was strongly associated with high VEGF/receptor expression in the vasculature. The high VEGF expression and the aMVD were not associated with the standard microvessel density (sMVD), as assessed with the monoclonal antibody anti-CD31 and, were not detected in normal tissue. An increased sMVD, however, was significantly related with the expression thymidine phosphorylase (TP), and also with the nuclear accumulation of the oncoprotein p53, but neither p53 nor TP was associated with VEGF expression by cancer cells or VEGF/receptor complex aMVD. In 35% of cancer cases examined, more than 20% of the microvessels assessed with anti-CD31 also expressed the VEGF/KDR complex. The vasculature of the normal head and neck mucosa did not express the VEGF/KDR complex. There was no association between VEGF expression or VEGF/receptor complex aMVD and response to chemo-radiotherapy or patient's survival. It is concluded that activation of the angiogenic pathway VEGF/flk-1(KDR) is tumor specific in a subgroup of locally advanced squamous cell carcinomas of the head and neck. Selective destruction of this type of vasculature, using immunoconjugates directed against the VEGF/receptor complex, may prove therapeutically useful for patients with a high tumoral VEGF/flk-1(KDR) activated microvessel fraction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Design of a variant of vascular endothelial growth factor-A (VEGF-A) antagonizing KDR/Flk-1 and Flt-1

Because of its central role in pathological angiogenesis, vascular endothelial growth factor (VEGF) has become a major target for anti-angiogenic therapies. We report here the construction of a heterodimeric antagonistic VEGF variant (HD-VEGF). In this antagonist, binding domains for the VEGF-receptors KDR/Flk-1 and Flt-1 are present at one pole of the dimer, whereas the other pole carries domain swap mutations, which prevent binding to either receptor. As HD-VEGF can only bind to monomeric receptors, it does not lead to signal transduction. Moreover, it antagonizes VEGF and possibly other members of the VEGF family, which are KDR/Flk-1 and Flt-1 ligands. We show here that HD-VEGF is a potent inhibitor of VEGF-mediated proliferation and tissue factor induction in endothelial cell cultures, requiring only a 20-fold and a 4-fold excess, respectively, to block the activity of wtVEGF completely. A 4-fold excess of HD-VEGF over wtVEGF was also sufficient to abrogate vascular permeability as determined in the Miles assay in vivo. Furthermore, HD-VEGF inhibited fetal bone angiogenesis in an ex vivo assay. Thus, HD-VEGF blocks KDR- and Flt-1-mediated VEGF activities that are crucial in the angiogenic process and is therefore a promising, multipotent compound in the treatment of angiogenesis-related diseases.     

Soluble Flt-1 regulates Flk-1 activation to control hematopoietic and endothelial development in an oxygen-responsive manner

Vascular endothelial growth factor (VEGF) and the vascular endothelial growth factor receptors (VEGFRs) regulate the development of hemogenic mesoderm. Oxygen concentration-mediated activation of hypoxia-inducible factor targets such as VEGF may serve as the molecular link between the microenvironment and mesoderm-derived blood and endothelial cell specification. We used controlled-oxygen microenvironments to manipulate the generation of hemogenic mesoderm and its derivatives from embryonic stem cells. Our studies revealed a novel role for soluble VEGFR1 (sFlt-1) in modulating hemogenic mesoderm fate between hematopoietic and endothelial cells. Parallel measurements of VEGF and VEGFRs demonstrated that sFlt-1 regulates VEGFR2 (Flk-1) activation in both a developmental-stage-dependent and oxygen-dependent manner. Early transient Flk-1 signaling occurred in hypoxia because of low levels of sFlt-1 and high levels of VEGF, yielding VEGF-dependent generation of hemogenic mesoderm. Sustained (or delayed) Flk-1 activation preferentially yielded hemogenic mesoderm-derived endothelial cells. In contrast, delayed (sFlt-1-mediated) inhibition of Flk-1 signaling resulted in hemogenic mesoderm-derived blood progenitor cells. Ex vivo analyses of primary mouse embryo-derived cells and analysis of transgenic mice secreting a Flt-1-Fc fusion protein (Fc, the region of an antibody which is constant and binds to receptors) support a hypothesis whereby microenvironmentally regulated blood and endothelial tissue specification is enabled by the temporally variant control of the levels of Flk-1 activation. Disclosure of potential conflicts of interest is found at the end of this article.     

蛋白激酶D1促血管新生的体内外实验分析

 目的: 探讨蛋白激酶D1(PKD1)促血管新生的作用,为心肌梗死等缺血性疾病以PKD1为治疗靶点提供新的思路。方法: 体外培养、分离和鉴定内皮祖细胞(EPCs),观察PKD1及其特异性阻断剂CID755673对EPCs中血管内皮生长因子(VEGF)及其受体KDR表达的影响。复制大鼠心肌梗死模型,分析PKD1及CID755673干预对大鼠心肌梗死后受损心肌组织病理形态学、微血管和内皮细胞变化以及VEGF、KDR表达的影响。结果: EPCs体外细胞培养实验表明,PKD1可明显上调EPCs中VEGF和KDR的表达水平。大鼠心肌梗死动物实验结果表明,PKD1干预后的大鼠心肌组织排列较为有序,结构较为清晰,内皮细胞胞膜光滑、完整,周细胞可见,心肌组织中的VEGF和KDR表达水平显著上调。结论: PKD1有明显的促血管新生作用,该作用可能是通过VEGF介导而实现的。     

抗KDR单链抗体的构建、表达及生物学活性鉴定

目的: 构建并表达抗人血管内皮生长因子受体KDR单链抗体(scFv)蛋白, 并测定其生物学活性。方法: 设计合成抗KDR单抗 (mAb)Ycom1D3VH、VL引物, 通过重叠延伸拼接 (splicingoverlapextensive, SOE)PCR, 在VH 和VL基因间引入柔性短肽(Gly4Ser)3, 构建抗KDRscFv基因并进行序列分析。将其克隆入pAYZH原核表达载体, 在大肠杆菌中诱导表达, 并以ELISA及免疫荧光结合实验测定重组蛋白的生物学活性。结果: 序列分析表明, 抗KDRscFv基因的全长为 729bp, 编码 243个氨基酸。将重组体表达产物进行SDS PAGE及Westernblot分析显示, 融合蛋白的相对分子质量(Mr)约为 30 000, 同预期的结果一致。表达产物主要以不溶性包涵体的形式存在, 表达量占菌体总蛋白的 20%。表达产物经变性、纯化及体外复性后, 纯度达 90%以上。ELISA和竞争性免疫荧光结合实验显示, 重组小分子scFv可与可溶性KDR抗原及表达KDR受体的人脐静脉内皮细胞特异性结合, 保留了亲本mAb的抗原结合活性。结论: 成功地构建了抗KDRscFv基因, 并在大肠杆菌中功能性表达, 为靶向诊断、治疗及进一步基因工程改造奠定了基础。     

Inhibition of avian leukosis virus replication by vector-based RNA interference

RNA interference (RNAi) has recently emerged as a promising antiviral technique in vertebrates. Although most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are more efficient and are practical for in vivo delivery. In this study, replication competent retroviral vectors were designed to deliver shRNA-mirs targeting subgroup B avian leukosis virus (ALV), the most effective of which reduced expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibited ALV(B) replication. This study demonstrates efficient antiviral RNAi in avian cells using shRNA-mirs expressed from pol II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline.     

Viral mediated gene transfer to sprouting blood vessels during angiogenesis

Several experimental systems have been applied to investigate the development of new blood vessels. Angiogenesis can be followed ex-vivo by culturing explants of rat aorta 'rings' in biomatrix gels. This angiogenesis system was modified for the study of viral vector mediated gene transfer, using adenovirus, vaccinia- and retroviral vectors. Two modifications were introduced to the model in order to facilitate efficient viral mediated gene transfer, (i) placing the aorta ring on top of a thin layer of collagen such that the angiogenic tissue will be accessible to the viral vector; and (ii) infection of the aorta rings prior to embedding them into the collagen matrix. While adenovirus and vaccinia vectors infected efficiently the aorta rings they induced cell death. Subsequent gene transfer experiments were, therefore, carried with retroviral vectors containing vascular endothelial growth factor (VEGF) and the beta-interferon (IFN) genes. Overexpression of VEGF enhanced significantly microvessel sprouting, while overexpression of IFN-beta induced an antiviral effect. The experimental system described in this study can facilitate the application of other viral vectors to the study of genes that may regulate the complex angiogenic process and thereby open new avenues for vascular gene therapy.     

Expression of the vascular endothelial growth factor receptor-2/Flk-1 in breast carcinomas: correlation with proliferation

Vascular endothelial growth factor (VEGF) and its receptor Flk-1/KDR play an important role in vascular permeability and tumor angiogenesis. Prompted by the hypothesis that VEGF/Flk-1 system may have regulatory roles in breast carcinogenesis, we investigated the expression of Flk-1 in 141 invasive breast carcinomas in correlation with clinical and immunohistochemical prognostic parameters, including proliferation indices like Ki-67 and Topoisomerase IIalpha (Topo-IIalpha). The immunohistochemical avidin-biotin-peroxidase method was performed on paraffin sections for the detection of Flk-1, p53, Bcl-2, c-erbB-2, Ki-67, Topo-IIalpha, ER, and PR. Flk-1 was detected in 91 of 141 (64.5%) of invasive breast carcinomas showing a widespread cytoplasmic expression in most of the neoplastic cells. Flk-1 expression was correlated with the menopausal status (P = 0.051) of the patient and the nuclear grade of the invasive breast carcinoma (P = 0.003), but demonstrated no correlation with histologic grade, stage, and patient survival. It is interesting that Flk-1 expression demonstrated a significant correlation with 2 well-established proliferation indices, Ki-67 (P = 0.037) and topo-IIalpha (P = 0.009), whereas there was no correlation with the expression of ER, PR, p53, Bcl-2, and c-erbB-2. Moreover, Flk-1 expression showed an inverse correlation with TIMP-1 mRNA localization in intratumoral stromal cells (P = 0.013). In conclusion, the significant correlation of Flk-1 expression in invasive breast carcinomas with proliferation indices like Ki-67 and topo-IIalpha suggests that VEGF may exert a growth factor activity on mammary cancer cells through its receptor Flk-1. On the other hand, the inverse correlation of Flk-1 with TIMP-1 mRNA in intratumoral stromal cells supports the notion that TIMP-1 may have an inhibitory role on angiogenesis.     

Targeting retrovirus to cancer cells expressing a mutant EGF receptor by insertion of a single chain antibody variable domain in the envelope glycoprotein receptor binding lobe

We have investigated targeting of retroviral vectors to a mutant EGF receptor (EGFRvIII) that is expressed in cancers of the brain, breast, lung and ovary, but is not found in any normal tissues. An expression plasmid was made in which a single chain Fv antibody specific for EGFRvIII was inserted at a novel position within a disulphide-bonded surface loop near the native receptor binding site of the Moloney leukemia virus ecotropic envelope glycoprotein. This fusion protein was expressed and incorporated into retroviral particles as efficiently as normal envelope glycoprotein. Retroviral vectors made with the fusion protein were able to bind peptide antigen and EGFRvIII expressed on the surface of human glioblastoma cells. The retroviral vectors had normal levels of infectivity on mouse cells, showing that the envelope glycoprotein tolerated a large insertion at this site, but did not show significant infectivity to human cells expressing EGFRvIII. Thus we were able to redirect retrovirus binding to this tumour-specific target without perturbing the normal function of the ecotropic envelope glycoprotein, but this was not sufficient to mediate infectivity via this receptor.     

Vascular endothelial growth factor-mediated autocrine stimulation of prostate tumor cells coincides with progression to a malignant phenotype

Vascular endothelial growth factor (VEGF), which is often produced at high levels by tumor cells, is a well-known mediator of tumor angiogenesis. VEGF receptor tyrosine kinases, KDR/Flk-1 and Flt-1, have been thought to be expressed exclusively by endothelial cells. In this study, we have used a prostate tumor progression series comprised of a differentiated rat prostate epithelial cell line, NbE-1, and its highly motile clonal derivative, FB2. Injection of NbE-1 cells into the inferior vena cava of syngeneic rats indicated that these cells are nontumorigenic. Using the same model, FB2 cells generated rapidly growing and well-vascularized tumors in the lungs. NbE-1 expressed marginal levels of VEGF, whereas high levels of VEGF protein were detected in FB2-conditioned medium and in FB2 tumors in vivo. Analysis of (125)I-VEGF(165) binding to NbE-1 and FB2 cells indicated that only motile FB2 cells expressed the VEGF receptor Flt-1. Consistent with this finding, physiological concentrations of VEGF induced chemotactic migration in FB2 but not in NbE-1 cells. This is the first documentation of a functional Flt-1 receptor in prostate tumor cells. Our results suggest two roles for VEGF in tumor progression: a paracrine role as an angiogenic factor and a previously undescribed role as an autocrine mediator of tumor cell motility.     

Novel highly efficient intrabody mediates complete inhibition of cell surface expression of the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR)

The human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) and its ligand vascular endothelial growth factor (VEGF) play an essential role in tumor angiogenesis and in haematological malignancies. To inhibit VEGF induced signalling, intrabodies derived from two scFv fragments recognizing the VEGF receptor were generated. When these intrabodies were expressed in endothelial cells, they blocked the transport of KDR to the cell surface. We developed a cell culture model using porcine aortic endothelial cells overexpressing KDR for testing the efficiency of anti-KDR intrabodies. The two intrabodies were targeted to the ER and colocalised with the KDR receptor in an intracellular compartment. No degradation of the receptor was observed. An immature incomplete glycosylated protein of 195 kDa was detected, suggesting that the intrabodies affect the maturation of the receptor. Despite the presence of significant amounts of receptor protein, the inactivation by one of the two intrabodies was highly effective, resulting in complete functional inhibition of KDR and inhibition of in vitro angiogenesis. The new intrabody appears to be a powerful tool with which to inhibit KDR function.     

Retroviral vector targeting through insertion of epidermal growth factor into receptor binding deficient influenza A hemagglutinin results in fusion defective particles

Targeting retroviral entry is a central theme in the development of vectors for gene therapy. The host range of a retrovirus is dependent upon the interaction of its envelope glycoprotein (Env) with a specific cell surface receptor protein, which allows viral entry. In contrast, the pH-dependent viruses enter cells through receptor-mediated endocytosis and the subsequent acidification produces conformational changes in the viral envelope protein(s) which lead to membrane fusion. We attempted to redirect retroviral vectors to epidermal growth factor (EGF) receptor expressing cells by using the pH-dependent influenza A virus hemagglutinin (HA). Wild type receptor binding was avoided either by point mutations or by deletion of the globular head structure of HA and also inserted EGF into HA. Replacement of the whole head domain was not tolerated. Two of the EGF-HA proteins bearing point mutations could be incorporated into retroviral particles, but unfortunately their fusion activity was lost. The data indicate that care must be taken when mutating multiple sites in HA, and that targeting HA requires further analysis of appropriate sites for the insertion of foreign sequences.