共查询到20条相似文献,搜索用时 15 毫秒
1.
Kirstin A Zettlitz Verena Lorenz Karlheinz Landauer Sabine Münkel Andreas Herrmann Peter Scheurich Klaus Pfizenmaier Roland E Kontermann 《MABS-AUSTIN》2010,2(6):639-647
Tumor necrosis factor (TNF) signals through TNFR1 and TNFR2, two membrane receptors, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB produced in CHO cells showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1–70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.Key words: humanized IgG, antagonistic antibody, tumor necrosis factor receptor 1, epitope mapping 相似文献
2.
《MABS-AUSTIN》2013,5(6):639-647
Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB, produced in CHO cells, showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates. 相似文献
3.
Rita Pellegrini Filippo Centis Stefania Martignone Antonio Mastroianni Elda Tagliabue Emanuela Tosi Sylvie Ménard Maria I. Colnaghi 《Cancer immunology, immunotherapy : CII》1991,34(1):37-42
Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 104 receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression. 相似文献
4.
Production and characterization of antibody blocking epidermal growth factor:receptor interactions 总被引:13,自引:0,他引:13
Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum. 相似文献
5.
6.
Receptor modulating properties of an antibody directed against the epidermal growth factor receptor 总被引:2,自引:0,他引:2
A murine antiserum with specificity for the human epidermal growth factor (EGF) receptor was used to investigate EGF receptor function. The IgG fraction of this antiserum displayed no EGF-like mitogenic activity, even when cross-linking was ensured by sequential treatment with rabbit anti-(mouse IgG). The interaction of antibody with solubilized purified EGF receptor was characterized in detail. The binding of 125I-antibody to the receptor was not blocked by EGF, but the binding of 125I-EGF to the receptor was blocked by the immune IgG. Scatchard analysis of this reaction revealed a reduction in maximal EGF binding but an enhanced EGF binding affinity. In addition, at low concentrations, the immune IgG was found to enhance receptor kinase activity in the absence of EGF. The enhancement of kinase activity, as measured by receptor phosphorylation, was due to a decreased Km for ATP, and an increased V. These results suggest that the antibody is capable of altering conformations at receptor active sites by binding to non-active species-specific epitopes. 相似文献
7.
Robert A. Boomsma Patricia A. Mavrogianis Harold G. Verhage 《Journal of molecular histology》1997,29(6):495-504
This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth
factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone
and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate
immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats.
Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation
animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was
used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated
animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors
was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor
was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation
sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast
was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of
pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all
the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal
growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play
an autocrine/paracrine role during reproduction 相似文献
8.
We have compared six different solutions commonly used for the extraction of peptides to optimize the extraction of Epidermal Growth Factor (EGF) from mouse submandibular glands. The yield of EGF was always higher in neutral pH extractions than in acidic ones. However, the EGF extracted at neutral pH was only poorly adsorbed on to octadecylsilyl silica, whereas the EGF extracted at acidic pH was be easily adsorbed. Subsequent purification of the EGF extracts by two-stage reversed-phase High Performance Liquid Chromatography yielded highly purified EGF. Identical chromatograms were obtained from each of the different extracts. Preparative scale quantities of EGF could be purified rapidly and reproducibly at high efficiency from acidic extracts. 相似文献
9.
K J Hyver J E Campana R J Cotter C Fenselau 《Biochemical and biophysical research communications》1985,130(3):1287-1293
Fast atom bombardment mass spectrometry has been used to characterize epidermal growth factor isolated from mouse submaxillary glands. The preparation is found to consist of two peptides, one of which has the average molecular weight predicted for the familiar gene product. The molecular weight of the second component is found to be reduced by the mass of one asparagine residue. These observations are discussed in light of previous reports of heterogeneity. 相似文献
10.
Garrido G Rabasa A Sánchez B López MV Blanco R López A Hernández DR Pérez R Fernández LE 《Journal of immunology (Baltimore, Md. : 1950)》2011,187(10):4954-4966
Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect. 相似文献
11.
Yoshida-Kato H Ichikawa K Yamaguchi J Watanabe K Ohsumi J Yonehara S Serizawa N 《Bioscience, biotechnology, and biochemistry》2000,64(9):1903-1908
Agonistic anti-human Fas antibodies that can induce apoptosis are thought to have therapeutic effects for various diseases resulting from an abnormality of the Fas/FasL system. However, some anti-Fas antibodies show toxicity, and it is difficult to investigate their therapeutic and toxicological effect using animals because of their species specificity. We previously obtained a murine anti-human Fas mAb, HFE7A. HFE7A reacted with both human and murine Fas, and mitigated lymphadenopathy without any sign of hepatotoxicity in MRLgld/gld mice. It is suggested that humanized HFE7A would be a therapeutic treatment for various diseases resulting from an abnormality of the Fas/FasL system. Here we isolated the cDNAs that code for the heavy and light chains of HFE7A and identified the corresponding nucleotide sequences. The recombinant HFE7A was indistinguishable in binding and apoptosis-inducing activity to that from a hybridoma cell line. These data provide essential information for the humanization and clinical application of the humanized HFE7A. 相似文献
12.
Structural studies have revealed two forms of the monomeric epidermal growth factor receptor (EGFR) ectodomain: a compact (tethered) form stabilized by interdomain interactions and an extended (untethered) form in the presence of ligand. An important question is whether the ligand induces a conformational transition from a tethered to untethered form or whether there is a preexisting conformational equilibrium between tethered and untethered states. To distinguish between these two possibilities, we investigated a truncated receptor, EGFR501 (spanning residues 1-501), that contains the minimal elements required for high-affinity ligand binding in solution. Conformational transitions and dynamics were inferred by means of fluorescence from five internal tryptophan residues that are located within or close to the ligand-binding domains of EGFR501. A preexisting conformational equilibrium between tethered and untethered states in EGFR501 was deduced from (1) the nonlinear Arrhenius temperature dependence of fluorescence and (2) fluorescence polarization showing independently mobile domains. In contrast, the ligand-EGFR501 complex revealed a linear Arrhenius temperature dependence of fluorescence and increased fluorescence polarization due to a lack of significant interdomain motions. The data suggest that the role of the ligand is to trap the EGFR501 in the untethered state that is transiently formed in solution through a preexisting conformational equilibrium. 相似文献
13.
Human, rat and mouse epidermal growth factors (EGF) bind to the same receptor on human placenta, but the binding characteristics differ. The apparent affinity constant (KA) for human EGF is higher (15 X 10(9) l/mol) than KA for rat EGF (10 X 10(9) l/mol). Mouse EGF binds with the lowest KA (5 X 10(9) l/mol). The pH optimum differs so that human and rat EGF bind with a pH optimum of 8.0, whereas mouse EGF binds with an optimum of pH 7.4. Half maximal dissociation is 130, 50 and 25 min for human, rat and mouse EGF, respectively. The structures of human, rat and mouse EGF differ somewhat. At least 11 of the first 24 residues differ. The N-terminal sequence of rat EGF is: Ala/Ser-Gly-X-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-X-Lys-Asp-Gly-Gly-Val-X-Met-Ty r-Val -Glu. 相似文献
14.
Identification of residues in the nucleotide binding site of the epidermal growth factor receptor/kinase 总被引:7,自引:0,他引:7
We have purified the epidermal growth factor receptor/kinase from A431 membrane vesicles which had been affinity labeled with the ATP analog, 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine. The resulting purified, affinity labeled receptor/kinase preparation has been subjected to reduction and carboxymethylation followed by tryptic digestion. From this digest, we have isolated and sequenced the tryptic peptide containing the major site of labeling by the ATP analog. The sequence of this peptide is Ile-Pro-Val-Ala-Ile-X-Glu-Leu, where X corresponds to Lys 721 of the derived sequence of the EGF receptor/kinase. 相似文献
15.
The epidermal growth factor receptor (EGFR) regulates key processes of cell biology, including proliferation, survival, and differentiation during development, tissue homeostasis, and tumorigenesis. Canonical EGFR activation involves the binding of seven peptide growth factors. These ligands are synthesized as transmembrane proteins comprising an N‐terminal extension, the EGF module, a short juxtamembrane stalk, a hydrophobic transmembrane domain, and a carboxy‐terminal fragment. The central structural and functional feature is the EGF module, a sequence containing six cysteines in a conserved spacement which is responsible for binding to the EGFR. While the membrane‐anchored peptide can be biologically active by juxtacrine signaling, in most cases the EGF module is proteolytically cleaved (a process termed ectodomain shedding) to release the soluble growth factor, which may act in an endocrine, paracrine, or autocrine fashion. This review summarizes the structural and functional properties of these fascinating molecules and presents selected examples to illustrate their roles in development, physiology, and pathology. J. Cell. Physiol. 218: 460–466, 2009. © 2008 Wiley‐Liss, Inc. 相似文献
16.
Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene 总被引:3,自引:0,他引:3
P Ray F J Moy G T Montelione J F Liu S A Narang H A Scheraga R Wu 《Biochemistry》1988,27(19):7289-7295
Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity. 相似文献
17.
J A Fernandez-Pol 《The Journal of biological chemistry》1985,260(8):5003-5011
A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells, denoted 2D1-IgM, was generated after fusion of immunized BALB/c mouse spleen cells with SP2/0-Ag14 myeloma cells. Specific binding of 2D1-IgM to the A431 cell-surface receptor for EGF was demonstrated by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Scatchard analysis of 125I-EGF binding to A431 cells demonstrated that 2D1-IgM treatment did not change the number of EGF receptors, but caused an increase in the affinity of EGF receptors from a population of low affinity to a uniform population of high affinity. Like EGF, 2D1-IgM induced phosphorylation of EGF receptors and EGF receptor clustering. As in the case of EGF, a biphasic growth response with stimulation of DNA synthesis at low and inhibition at high concentrations of 2D1-IgM was evident in A431 cells. The intrinsic "EGF-like" bioactivity of 2D1-IgM was enhanced by the presence of EGF. These results suggest that the binding of 2D1-IgM to the EGF receptor at a different site from that to which EGF binds can initiate an effective EGF-like biological response; and the EGF-like biological effects of 2D1-IgM may be mediated by a population of high affinity EGF receptors which may be involved in the control of cellular growth. 相似文献
18.
Tyrosine residues in bovine phospholipase C-gamma phosphorylated by the epidermal growth factor receptor in vitro. 总被引:13,自引:0,他引:13
J W Kim S S Sim U H Kim S Nishibe M I Wahl G Carpenter S G Rhee 《The Journal of biological chemistry》1990,265(7):3940-3943
We have identified the sites phosphorylated in vitro by epidermal growth factor (EGF) receptor kinase in bovine brain phospholipase C-gamma (PLC-gamma). They are tyrosine residues 472, 771, 783, and 1254. The rate of phosphorylation was fastest with the sites at 771 and 783, then at 1254, and slowest at 472. PLC-gamma isolated from cells treated with EGF is known to contain at least four tyrosine phosphate-containing peptides and two of them are identified to be residues 771 and 1254 in the accompanying paper (Wahl, M. I., Nishibe, S., Kim, J. W., Kim, H., Rhee, S. G., and Carpenter, G. (1990) J. Biol. Chem. 265, 3944-3948). The 3 residues 472, 771, and 783 are located closely to the regions of PLC-gamma which exhibit a high sequence similarity to the regulatory domain of the src family tyrosine kinases. Nevertheless, the tyrosine phosphorylation did not affect the catalytic activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by EGF receptor kinase alters its interaction with putative inhibitory proteins and leads to its activation. 相似文献
19.
Klaus Koefoed Lucilla Steinaa Josefine Nielsen S?derberg Ida Kj?r Helle Jane Jacobsen Per-Johan Meijer John S?rensen Haurum Allan Jensen Michael Kragh Peter Sejer Andersen Mikkel Wandahl Pedersen 《MABS-AUSTIN》2011,3(6):584-595
The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.Key words: EGFR, antibody synergy, functional screening, epitope binning, antibody combinations 相似文献
20.
《MABS-AUSTIN》2013,5(6):584-595
The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation. 相似文献