Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method

We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of "target-guided proximity labeling," such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100? from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 ?. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1 × 1012 pfu, 47 each of Biotin-labeled scFv-displaying phages were recovered using Streptoavidin-coated magnetic beads, and among them total 11 scFv-phages were found to be definitely positive for binding to A431 cell surface by ELISA assay. Restriction mapping and sequencing analysis of these scFv-phage DNAs revealed that they encode 4 different scFv-nucleotide sequences in total. Immuno-fluorescent microscopy provided evidence that these 4 scFv antibodies bind specifically to EGFR on the A431 cells, showing slightly different staining patterns. Thus, "target-guided proximity labeling" methods were powerful for isolating scFv-displaying phages that recognize distinct extracellular domains of the target receptor. This novel screening strategy could be applicable to many other cell surface antigens and receptors.     

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope

One of the proposed approaches in cancer therapy is to induce and direct the patient’s own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.     

Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies

To generate a panel of antibodies binding human breast cancers, a human single chain Fv phage display library was selected for rapid internalization into the SK-BR-3 breast cancer cell line. Thirteen unique antibodies were identified within the 55 cell binding antibodies studied, all of them showing specific staining of tumor cells compare to normal epithelial cells. Two of the antibodies bound the ErbB2 oncogene while 6 bound the tumor marker transferrin receptor (TfR). By developing a scFv immunoprecipitation method, we were able to use LC-MS/MS to identify the antigen bound by one of the antibodies (3GA5) as FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1 (CD9 partner 1) an Ig superfamily member that has been described to interact directly with CD9 and CD81 tetraspanins and to be overexpressed in adherent cancer cell lines. Although the 3GA5 scFv had no direct anti-proliferative effect, intracellular expression of the scFv was able to knockdown CD9P-1 expression and could be used to further define the role of the tetraspanin system in proliferation and metastasis. Moreover, the 3GA5 scFv was rapidly internalized into breast tumor cells and could have potential for the targeted delivery of cytotoxic agents to breast cancers. This study is the proof of principle that the direct selection of phage antibody libraries on tumor cells can effectively lead to the identification and functional characterization of relevant tumor markers.     

Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.     

Displaying epidermal growth factor on spleen necrosis virus-derived targeting vectors

Targeted gene transfer into human cells has previously been achieved with spleen necrosis virus (SNV)-derived vector particles harboring envelope (Env) proteins which carry single chain Fv (scFv) domains derived from antibodies. Such cell targeting vectors have been found to directly transduce human cells expressing the cell surface molecules recognized by the respective scFv. In an attempt to achieve targeted gene transfer into epidermal growth factor receptor (EGFR)-positive human cells, SNV vector particles carrying a surface (SU) envelope protein N-terminally modified with the EGF domain and the wildtype transmembrane protein were generated. However, direct transduction of EGFR-positive cells was not detected. Canine D17 cells, which can be infected by wildtype SNV, were also not transduced. Infectivity of D17 cells was restored by removal of the EGF modification via cleavage of a factor Xa site located between the EGF domain and the SU protein or by blocking the EGFRs on the cell surface by EGF treatment. The properties of SNV-EGF vector particles as described here are similar to those of murine leukemia virus-derived vector particles harboring envelope proteins modified with a growth factor-derived domain. It seems therefore that, although scFv-modified SNV allows direct cell targeting, EGF-modified SNV allows only indirect cell targeting.     

Overexpression of Thrombospondin-1 Decreases Angiogenesis and Inhibits the Growth of Human Cutaneous Squamous Cell Carcinomas

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in epithelial tumor development has remained controversial. We studied the in vitro growth characteristics and the in vivo tumor xenograft growth of the human squamous cell carcinoma cell lines A431 and SCC-13, stably transfected to overexpress human TSP-1. Overexpression of TSP-1 inhibited tumor growth of A431 xenotransplants, and completely abolished tumor formation by SCC-13 cells. TSP-1 overexpressing A431 tumors were characterized by extensive areas of necrosis and by decreased tumor vessel number and size. The effects of TSP-1 on tumor cell growth were indirect since tumor cell proliferation rates in vivo and in vitro, anchorage-dependent and -independent growth in vitro, and susceptibility to induction of apoptosis by serum withdrawal were unchanged in TSP-1 overexpressing tumor cells. However, TSP-1 overexpression up-regulated the TSP-1 receptor CD36, leading to enhanced adhesion of A431 cells to TSP-1. These findings establish TSP-1 as a potent inhibitor of angiogenesis and tumor growth in carcinomas of the skin.     

Isolation and characterization of human antibodies targeting human aspartyl (asparaginyl) beta-hydroxylase

Over-expression of the enzyme human aspartyl (asparaginyl) beta-hydroxylase (HAAH) has been detected in a variety of cancers. It is proposed that upon cellular transformation, HAAH is overexpressed and translocated to the tumor cell surface, rendering it a specific surface antigen for tumor cells. In this work, twelve human single-chain Fv fragments (scFv) against HAAH were isolated from a human non-immune scFv library displayed on the surface of yeast. Five of the twelve were reformatted as human IgG1. Two of the five IgGs, 6-22 and 6-23, showed significant binding to recombinant HAAH in ELISA, tumor cell lines, and tumor tissues. The apparent dissociation constants of 6-22 and 6-23 IgG were 1.0 +/- 0.2 nM and 20 +/- 10 nM respectively. These two antibodies were shown to target different domains of HAAH, with 6-22 targeting the catalytic domain of HAAH and 6-23 targeting the N-terminal non-catalytic domain of HAAH. 6-22 IgG was further characterized, as it had high affinity and targeted the catalytic domain. 6-22 IgG alone does not exhibit significant cytotoxicity toward the tumor cells. However, 6-22 internalizes into tumor cells and can therefore be employed to deliver cytotoxic moieties. A goat anti-human IgG-saporin conjugate was delivered into tumor cells by 6-22 IgG and hence elicited cytotoxicity toward the tumor cells in vitro. These tumor-binding human antibodies can potentially be used in both diagnosis and immunotherapy targeting HAAH-expressing tumor cells.     

The use of intracellular single-chain antibody fragments to inhibit specifically the expression of cell surface molecules

One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum-retained, single chain Fv antibody fragments (scFv) were investigated. These intracellular scFv constructs were tested for their ability to inhibit specifically the expression of a CHO cell line pretransfected with the relevant cell surface antigen CD2. No scFv was detected in the cell supernatant although functional scFv, as assayed by ELISA, was detected in an NP-40 soluble fraction if an N-linked glycosylation site had been introduced into the antibody construct. This demonstrates that functional antibody combining sites can be produced even though they are not secreted. Inhibition of CD2 was obtained but was not complete and differed between clones. Levels of scFv could be increased by gene amplification but the level of functional binding activity remained constant and no further inhibition of CD2 expression was obtained. Immunofluorescence analysis at the single-cell level of the permeabilised transfected cell lines showed that less than 8% of cells expressed detectable levels of intracellular scFv, indicating selection against cells producing high levels of single-chain antibody. This selection was not seen when comparable single-chain TCR constructs, known to be retained intracellularly, were used. Thus, production of scFv with binding activity is not sufficient for good inhibition of gene expression although introduction of an N-linked glycosylation site is beneficial. The best strategy is probably to screen a panel of scFv constructs and use those that are secreted rather than those that are retained intracellularly     

Selection of Potential Therapeutic Human Single-Chain Fv Antibodies against Cholecystokinin-B/Gastrin Receptor by Phage Display Technology

Background and Objective

Gastric/gastrointestinal cancers are associated with high mortality worldwide. G-protein coupled receptor (GPCR) superfamily members such as gastrin/cholecystokinin-B receptor (CCK-BR) are involved in progression of gastric tumors, thus CCK-BR is considered as a potential target for immunotherapy. However, production of functional monoclonal antibodies (mAbs) against GPCR seems to be very challenging, in part due to its integration in cell membranes and inaccessibility for selection. To tackle this problem, we implemented phage display technology and a solution-phase biopanning (SPB) scheme for production of mAbs specific to the native conformation of CCK-BR.

Methods

To perform the SPB process, we utilized a synthetic biotinylated peptide corresponding to the second extracellular loop (ECL2) of CCK-BR and a semi-synthetic phage antibody library. After enzyme-linked immunosorbent assay (ELISA) screening, the CCK-BR specificity of the selected single-chain variable fragments (scFvs) were further examined using immunoblotting, whole-cell ELISA, and flow cytometry assays.

Results

After performing four rounds of selection, we identified nine antibody clones which showed positive reactivity with the CCK-BR peptide in an ELISA assay. Of these, eight clones were unique scFv antibodies and one was a V_L single domain antibody. Specificity analysis of the selected scFvs revealed that five of the selected scFvs recognized a denatured form of CCK-BR, while the majority of the selected scFvs were able to recognize the native conformation of CCK-BR on the surface of human gastric adenocarcinoma cells and cervical carcinoma HeLa cells.

Conclusion

For the first time, we report on the establishment of a diverse panel of scFv antibody fragments that are specific to the native conformation of CCK-BR. Based on these results, we suggest the selected scFv antibody fragments as potential agents for diagnosis, imaging, targeting, and/or immunotherapy of cancers that overexpress CCK-BR.     

Global protein profiling reveals anti-EGFR monoclonal antibody 806-modulated proteins in A431 tumor xenografts

Abstract

An important mediator of tumorigenesis, the epidermal growth factor receptor (EGFR) is expressed in almost all non-transformed cell types, associated with tumor progression, angiogenesis and metastasis. The significance of the EGFR as a cancer therapeutic target is underscored by the clinical development of several different classes of EGFR antagonists, including monoclonal antibodies (mAb) and tyrosine kinase inhibitors. Extensive preclinical studies have demonstrated the anti-tumor effects of mAb806 against tumor xenografts overexpressing EGFR. EGF stimulation of A431 cells induces rapid tyrosine phosphorylation of intracellular signalling proteins which regulate cell proliferation and apoptosis. Detailed understanding of the intracellular signalling pathways and components modulated by mAbs (such as mAb806) to EGFR, and other growth factor receptors, remain limited. The use of fluorescence 2D difference gel electrophoresis (2D DIGE), coupled with sensitive MS-based protein profiling in A431 tumor (epidermoid carcinoma) xenografts, in combination with mAb806, revealed proteins modulating endocytosis, cell architecture, apoptosis, cell signalling pathways and cell cycle regulation, including Dynamin-1-like protein, cofilin-1 protein, and 14-3-3 protein zeta/delta. Further, we report various proteins, including Interferon-induced protein 53 (IFI53), and Oncogene EMS1 (EMS1) which have roles in the tumor microenvironment, regulating cancer cell invasiveness, angiogenesis and formation of metastases. These findings contribute to understanding the underlying biological processes associated with mAb806 therapy of EGFR-positive tumors, and identifying further potential protein markers that may contribute in assessment of the treatment response.     

T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo

T cells modified with chimeric antigen receptor are an attractive strategy to treat Epstein-Barr virus（EBV） associated malignancies.The EBV latent membrane protein 1（LMP1） is a 66-KD integral membrane protein encoded by EBV that consists of transmembrane-spanning loops.Previously,we have identified a functional signal chain variable fragment（scFv） that specifically recognizes LMP1 through phage library screening.Here,we constructed a LMP1 specific chimeric antigen receptor containing anti-LMP1 scFv,the CD28 signalling domain,and the CD3ζchain（HELA/CAR）.We tested its functional ability to target LMP1 positive nasopharyngeal carcinoma cells.HELA/CAR cells were efficiently generated using lentivirus vector encoding the LMP1-specific chimeric antigen receptor to infect activated human CD3＋ T cells.The HELA/CAR T cells displayed LMP1 specific cytolytic action and produced IFN-γ and IL-2 in response to nasopharyngeal carcinoma cells overexpressing LMP1.To demonstrate in vivo anti-tumor activity,we tested the HELA/CAR T cells in a xenograft model using an LMP1 overexpressing tumor.Intratumoral injection of anti-LMP1 HELA/CAR-T cells significantly reduced tumor growth in vivo.These results show that targeting LMP1 using HELA/CAR cells could represent an alternative therapeutic approach for patients with EBV-positive cancers.     

Assembly of targeting complexes driven by a single-chain antibody

Rapid development in design and production of recombinant antibodies and antibody fragments specific for cell surface markers opens new opportunities for targeted delivery of therapeutic or imaging agents. However, the progress in this field is slowed by inactivation of many antibodies by chemical conjugation of payloads and by lack of internalization of complexes formed on the cell surface. Here, we describe conversion of a non-internalizing single chain Fv (scFv) antibody P4G7 specific for vascular endothelial growth factor receptor 2 (VEGFR-2) into a targeting protein (Hu-P4G7) for assembly of a novel type of targeting complexes. Hu-P4G7 contains an N-terminal "docking" Hu-tag, a 15-aa fragment of human RNase I, capable of high affinity binding of S-protein fragment of human RNase I or bovine RNase A. Purified Hu-P4G7 and complexes of Hu-P4G7 with S-protein bind both soluble and full-length cellular VEGFR-2. To assemble targeted DNA delivery complexes, S-protein modified with a DNA condensing agent was "docked" to Hu-P4G7, and then loaded with luciferase plasmid DNA. As expected for a non-internalizing targeting protein, Hu-P4G7-based complexes did not deliver DNA in VEGFR-2 expressing cells. However, in the presence of vascular endothelial growth factor (VEGF), these complexes selectively delivered DNA into the cells overexpressing VEGFR-2 suggesting that even a non-internalizing scFv antibody can be used for targeted intracellular drug delivery.     

Recombinant full-length human IgG1s targeting hormone-refractory prostate cancer

We have used a naive human single-chain fragment variable (scFv) library as a source of random shape repertoire to directly probe the altered surface chemistry of tumor cells. We reported previously the identification of more than 90 internalizing phage monoclonal antibodies targeting prostate cancer cells, including those that are hormone refractory. In this report, we describe the conversion of a panel of those scFvs into full-length human immunoglobulins (IgGs) and show that tumor specificity is retained. We have further shown that antibodies isolated from a naive phage display library can nevertheless be of high affinity towards target tumor cells. In addition, full-length IgGs retain the functionality of parental scFvs including the ability to rapidly enter target cells through receptor-mediated endocytosis and thereby to mediate efficient and specific intracellular payload delivery to tumor cells. We have used recombinant IgGs to immunoprecipitate target antigens and analyzed their molecular composition by mass spectrometry. We have identified one target antigen as activated leukocyte cell adhesion molecule (ALCAM)/MEMD/CD166 and have further studied tissue specificity of this internalizing ALCAM epitope by immunohistochemistry. Our study shows that cell type-specific internalizing human antibody can be readily identified from a naive phage antibody display library, characterized with regards to sequence, affinity, tissue specificity, and antigen identity, and modified genetically and chemically to generate various forms of targeted therapeutics.     

抗体-白细胞介素2融合蛋白183B2scFv-Interleukin 2的表达及活性检测

目的在哺乳动物细胞内表达抗体细胞因子融合蛋白183B2scFv Interleukin2,为卵巢癌提供一种新的治疗方法。方法通过基因工程的方法将两段基因IL2和183B2scFv开放读框的编码序列克隆在一起,在CHO K1细胞内人巨细胞病毒启动子的作用下表达可溶性融合蛋白,并检测其对肿瘤细胞的靶向性。结果采用哺乳动物细胞表达,系统表达的这种小分子融合蛋白,既保留了与卵巢癌OC183B2抗原结合的特性,又保持了IL2的生物学活性。融合蛋白在细胞培养上清中保持稳定,更重要的是融合蛋白将IL2靶向表达OC183B2抗原的卵巢癌细胞表面化的同时,又能刺激IL2依赖细胞株的增殖,从而诱发局部有效的抗肿瘤反应。既提高了融合蛋白的穿透组织能力和肿瘤组织部位的浓聚,又避免了大剂量全身应用细胞因子引起的副作用。结论真核表达系统表达的融合蛋白具有很好的生物学活性。     

Production and characterization of monoclonal antibodies to pituitary adenylate cyclase activating polypeptide type I receptor

Pituitary adenylate cyclase activating polypeptide type I receptor (PACAPr) belongs to the novel subfamily of the G-protein coupled receptors with a long extracellular N-terminus, which functions as a major binding site for the PACAP. Three different N-terminal fragments of rat PACAPr were overexpressed in Escherichia coli and purified using His-tags or maltose-binding protein as anchors for affinity chromatography. The purified and refolded proteins were used for the production and screening of monoclonal antibodies (MAbs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PACAPr were generated and characterized. Epitope analysis by competitive enzyme-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity of MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immunoblotting and flow cytometric analysis using transiently transfected COS cells and stably transfected CHO cells expressing rat PACAPr. Each antibody was examined by immunoblotting for the ability to cross react with the human PACAPr in human neuroblastoma NB-OK cells and most of them were shown to recognize human PACAPr as effectively as rat PACAPr. MAbs against the N-terminal extracellular domain of PACAPr can be used for the immunochemical study of the receptor-ligand interaction and for the investigation of PACAPr distribution in normal and tumor tissues.     

Mechanism of HSV infection through soluble adapter-mediated virus bridging to the EGF receptor

Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.     

EGFR and ErbB2 differentially regulate Raf-1 translocation and activation.

Epidermal growth factor receptor (EGFR) and HER-2/ErbB2 are members of the Erb family of signaling receptors. ErbB2 is overexpressed in many different cancers and has been linked to enhanced malignancy of tumors. We have examined the cellular translocation of Raf-1 during EGF-dependent signal transduction in two breast tumor cell lines, BT20 and SKBR3. Treatment of BT-20 breast cancer cells, which express EGFR, with EGF resulted in rapid (5 minutes) accumulation of EGFR and Raf-1 into plasma membrane-associated endocytotic vesicles. However, at later time points (30 minutes) only EGFR was endocytosed and Raf-1 dissociated from the plasma membrane and was found in the cytosol. In SKBR3 breast cancer cells, which express high levels of EGFR and ErbB2, treatment with EGF also resulted in rapid accumulation of EGFR and Raf-1 into endocytotic vesicles, but EGFR endocytosis was inhibited and Raf-1 remained associated with the plasma membrane for a prolonged period. The role of ErbB2 in the retention of Raf-1 at the plasma membrane was confirmed in BT-20 cells transfected with ErbB2. BT-20 cells expressing ErbB2 and treated with EGF retained Raf-1 at the plasma membrane for prolonged periods, whereas Raf-1 rapidly dissociated from the plasma membrane in EGF-stimulated cells transfected with a control vector. The presence of Raf-1 at the plasma membrane correlated with activation of Raf-1 and MAP kinase. Cells that expressed ErbB2 and treated with EGF showed prolonged activation of Raf-1 and MAP kinase compared with cells that expressed low levels of ErbB2. These results suggest that expression of ErbB2 promoted retention of Raf-1 in the plasma membrane, resulting in prolonged activation of the MAP kinase cascade, which may contribute to enhanced malignancy in ErbB2-expressing cancers.     

Murine endoglin-specific single-chain Fv fragments for the analysis of vascular targeting strategies in mice

Endoglin has been identified as a promising cell surface antigen for vascular targeting approaches in cancer therapy, e.g. employing antibody molecules as targeting moieties. However, in vivo analysis of such strategies in mouse models requires antibodies recognizing endoglin on mouse endothelial cells. Here we describe the isolation of single-chain Fv fragments (scFvs) from phage display libraries, which bind to the extracellular region of mouse endoglin. One of these clones, scFv mE12, showed strong (K_d = 11 nM) and selective binding to purified endoglin and also to the endoglin-expressing mouse endothelioma cell line eEnd.2. This antibody recognized a linear epitope located in the N-terminal region (aa 27-361) of endoglin. Cell binding was further increased by generating a bivalent scFv-Fc fusion protein composed of scFv mE12 and the human γ1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes capable of selectively binding to endoglin-expressing cells. Thus, anti-mouse endoglin scFv mE12 should be useful to analyze vascular targeting strategies in mice.     

Model systems to study the parameters determining the success of phage antibody selections on complex antigens

Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen". We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.     

CD95-mediated Apoptosis of Human Glioma Cells: Modulation by Epidermal Growth Factor Receptor Activity

The death ligands CD95L and Apo2L/TRAIL are promising investigational agents for the treatment of malignant glioma. EGFR is overexpressed in a significant proportion of malignant gliomas in vivo. Here, we report that CD95L-induced cell death is enhanced by EGFR inhibition using tyrphostine AG1478 in 7 of 12 human malignant glioma cell lines. Conversely, CD95-mediated and Apo2L-induced cell death are both inhibited by overexpression of EGFR in LN-229 cells. CD95L-induced cell death augmented by AG1478 is accompanied by enhanced processing of caspase 8. LN-229 cells overexpressing the viral caspase inhibitor, crm-A, are not sensitized to CD95L-induced cell death by AG1478, indicating that EGFR exerts its antiapoptotic properties through a caspase 8-dependent pathway. These data define a modulatory effect of EGFR-activity on death ligand-induced apoptosis and indicate that EGFR inhibition is likely to improve the efficacy of death ligand-based cancer therapies. Furthermore, it is tempting to speculate that EGFR amplification protects tumor cells from death ligand-mediated host immune responses in vivo and that EGFR's effects on death receptor-mediated apoptosis may explain the anti-tumor effects of non-cytotoxic, unarmed anti-EGFR family antibodies.