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1.
Yip YL Smith G Koch J Dübel S Ward RL 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(8):5271-5278
The self-oncoprotein ErbB-2 is overexpressed in a number of malignancies. The presence of endogenous anti-ErbB-2 Ab and T cell immune responses to this protein in cancer patients has made ErbB-2 an attractive target for active immunization. However, the finding that murine anti-ErbB-2 Abs can have stimulatory, inhibitory, or no effects on cancer cell growth suggests that an inappropriately induced immune response may have an adverse effect. To ensure the induction of a beneficial Ab response, it is important to identify the epitopes recognized by these Abs. In this study we have used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-ErbB-2 mAbs. The epitopes of three mAbs, N12, N28, and L87, were successfully located to C531-A586, T216-C235, and C220-C235 of ErbB-2, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines overexpressing ErbB-2. The peptide region recognized by N12, (C531-A586; EP531), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide (GST-EP531) recognized the peptide region EP531 as well as native ErbB-2. More importantly, Igs purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. In conclusion, our study provides direct evidence of the function-epitope relationship of anti-ErbB-2 Abs and also emphasizes the value of inducing a potent tumor inhibitory polyclonal Ab response by rationally selecting regions of ErbB-2 used for immunization. 相似文献
2.
S A Pilon M P Piechocki W Z Wei 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(6):3201-3206
Wild-type ErbB-2 (E2) positive D2F2/E2 tumors are rejected by active vaccination with ErbB-2 DNA. However, anti-ErbB-2 Ab response can cause cardiac toxicity or interfere with cellular immunity. It will be advantageous to induce only cellular immunity by active vaccination. A panel of E2 DNA vaccines were constructed, and their vaccination efficacy was ranked as E2 > tyrosine kinase-deficient ErbB-2 (E2A) > full-length ErbB-2 targeted to the cytoplasm (cytE2) > tyrosine kinase-deficient cytE2 (cytE2A). E2A is a tyrosine kinase-deficient mutant containing a single residue substitution. CytE2 or cytE2A encodes a full-length protein that is targeted to and rapidly degraded in the cytosol by the proteasomes. Covaccination with cytE2A and GM-CSF or IL-2 DNA resulted in equivalent anti-tumor activity as E2. However, anti-ErbB-2 Ab was induced by E2 or E2A, but not cytE2 or cytE2A. Therefore, cytE2A appears to induce anti-tumor immunity without an Ab response. ErbB-2-specific CTL were detected in mice immunized with cytE2A and GM-CSF and have rejected tumor challenge. Depletion of CD8, but not CD4 T cells reduced anti-tumor immunity, indicating CTL as the effector cells. Covaccination with E2A and cytE2A induced synergistic anti-tumor activity, supporting enhanced peptide presentation from cytE2A, which was further evidenced by superior CTL activation using APCs expressing cytE2 vs E2. Taken together, cytoplasmic ErbB-2 DNA induced anti-tumor CTL, but not humoral response, demonstrating the feasibility of eliciting individual effector mechanism by targeted DNA vaccine. 相似文献
3.
Kim SO Houtman JC Jiang J Ruppert JM Bertics PJ Frank SJ 《The Journal of biological chemistry》1999,274(50):36015-36024
The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status. 相似文献
4.
De Luca A Carotenuto A Rachiglio A Gallo M Maiello MR Aldinucci D Pinto A Normanno N 《Journal of cellular physiology》2008,214(3):559-567
The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB-2, ErbB-3, and ErbB-4) that are activated following binding to epidermal growth factor (EGF)-like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non-malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB-2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti-EGFR or anti-ErbB-2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor-associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth. 相似文献
5.
Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2-mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse. 相似文献
6.
Suzuki M Shinkai M Honda H Kamihira M Iijima S Kobayashi T 《Biochimica et biophysica acta》2001,1525(1-2):191-196
Cells expressing a membrane-anchored single-chain fragment variable (scFv) domain against a tumor-specific antibody were fabricated. These cells were able to bind to cells of a human colon cancer line (BM314) expressing the erbB-2 proto-oncogene. A plasmid, pMFverbB, was first constructed in which the anti-ErbB-2 scFv gene was cloned in-frame between a signal peptide sequence and the platelet-derived growth factor receptor (PDGFR) transmembrane domain gene to express scFv on the cell surface. An African green monkey cell line, COS-1, was stably transfected with pMFverbB. Immunofluorescence assay experiments and microscopic observation showed that the cells expressing scFv bound to the human tumor cells overexpressing the ErbB-2 protein as well as to cells of a mouse fibroblast line (NIH-3T3) transfected with the erbB-2 gene. The cells expressing scFv could take up magnetite cationic liposomes as a model of particle-type drug and retained the ability to target ErbB-2-expressing cells. The fabricated cells have the potential to serve as drug carriers in drug targeting applications. 相似文献
7.
Itoh K Inoue K Tezuka T Tada H Hashimoto Y Masuko T Suzuki T 《Journal of biochemistry》2003,133(2):239-245
To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2. 相似文献
8.
ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner 总被引:13,自引:2,他引:11
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Monilola A. Olayioye Diana Graus-Porta Roger R. Beerli Jack Rohrer Brigitte Gay Nancy E. Hynes 《Molecular and cellular biology》1998,18(9):5042-5051
The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed. 相似文献
9.
The transmembrane domains of ErbB receptor tyrosine kinases are monotopic helical structures proposed to be capable of direct side-to-side contact with related receptors. Formation of the resulting homo- or hetero-oligomeric complexes is considered a key step in ligand-mediated signalling. ErbB-2, which has not been observed to form active homo-dimers in a ligand dependent manner, has been implicated as an important partner for formation of hetero-dimers with other ErbB receptors. Recent work has shown that the ErbB-2 transmembrane domain is capable of forming homo-oligomeric species in lipid bilayers, while a similar domain from ErbB-1 appears to have a lesser tendency to such interactions. Here, 2H nuclear magnetic resonance was used to investigate the role of the ErbB-2 transmembrane domain in hetero-oligomerisation with that of ErbB-1. At low total concentrations of peptide in the membrane, ErbB-2 transmembrane domains were found to decrease the mobility of corresponding ErbB-1 domains. The results are consistent with the existence of direct transmembrane domain involvement in hetero-oligomer formation within the ErbB receptor family. 相似文献
10.
Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library 总被引:1,自引:0,他引:1
Karasseva NG Glinsky VV Chen NX Komatireddy R Quinn TP 《Journal of Protein Chemistry》2002,21(4):287-296
The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 M. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor. 相似文献
11.
12.
ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes. 相似文献
13.
14.
Stortelers C van De Poll ML Lenferink AE Gadellaa MM van Zoelen C van Zoelen EJ 《Biochemistry》2002,41(13):4292-4301
Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members. 相似文献
15.
Labriola L Salatino M Proietti CJ Pecci A Coso OA Kornblihtt AR Charreau EH Elizalde PV 《Molecular and cellular biology》2003,23(3):1095-1111
The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestin-dependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro. 相似文献
16.
Single-chain antibody-mediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling. 总被引:22,自引:8,他引:14
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ErbB-2 becomes rapidly phosphorylated and activated following treatment of many cell lines with epidermal growth factor (EGF) or Neu differentiation factor (NDF). However, these factors do not directly bind ErbB-2, and its activation is likely to be mediated via transmodulation by other members of the type I/EGF receptor (EGFR)-related family of receptor tyrosine kinases. The precise role of ErbB-2 in the transduction of the signals elicited by EGF and NDF is unclear. We have used a novel approach to study the role of ErbB-2 in signaling through this family of receptors. An ErbB-2-specific single-chain antibody, designed to prevent transit through the endoplasmic reticulum and cell surface localization of ErbB-2, has been expressed in T47D mammary carcinoma cells, which express all four known members of the EGFR family. We show that cell surface expression of ErbB-2 was selectively suppressed in these cells and that the activation of the mitogen-activated protein kinase pathway and p70/p85S6K, induction of c-fos expression, and stimulation of growth by NDF were dramatically impaired. Activation of mitogen-activated protein kinase and p70/p85S6K and induction of c-fos expression by EGF were also significantly reduced. We conclude that in T47D cells, ErbB-2 is a major NDF signal transducer and a potentiator of the EGF signal. Thus, our observations demonstrate that ErbB-2 plays a central role in the type I/EGFR-related family of receptors and that receptor transmodulation represents a crucial step in growth factor signaling. 相似文献
17.
《Cell cycle (Georgetown, Tex.)》2013,12(15):1946-1950
Pheochromocytoma (PCC) is a rare catecholamine-producing tumor that arises from the adrenal medulla and is often familial. The genetic basis for familial PCC involves mutations of RET, VHL, SHDx or NF-1 in more than 20% of cases. Additional genes may be important in pathogenesis of both familial and sporadic PCC. ErbB-2/Her2/Neu is a growth factor receptor tyrosine kinase that is frequently overexpressed in tumors and there is clinical evidence suggesting that enhanced ErbB-2 growth factor receptor signaling may play a role in PCC. In the present study, ectopic expression of an activated ErbB-2 transgene resulted in bilateral adrenal PCC. Analyses of tumor samples and normal adrenal tissue revealed that levels of the Pten tumor suppressor protein were greatly reduced in PCCs, while levels of the cell cycle regulatory protein cyclin D1 were usually increased. In addition, levels of phospo-AKT were increased in PCCs versus normal adrenal tissue. Biochemical analyses established that PCC’s were functionally active, producing abundant levels of the catecholamines, epinephrine and norepinephrine. These data establish that increased ErbB-2 growth factor receptor signaling in the adrenal medulla can lead to PCC through combined influences on Pten, AKT and cyclin 相似文献
18.
The deaf and the dumb: the biology of ErbB-2 and ErbB-3 总被引:22,自引:0,他引:22
ErbB-2 (also called HER2/neu) and ErbB-3 are closely related to the epidermal growth factor receptor (EGFR/ErbB-1), but unlike EGFR, ErbB-2 is a ligandless receptor, whereas ErbB-3 lacks tyrosine kinase activity. Hence, both ErbB-2 and ErbB-3 are active only in the context of ErbB heterodimers, and ErbB-2. ErbB-3 heterodimers, which are driven by neuregulin ligands, are the most prevalent and potent complexes. These stringently controlled heterodimers are repeatedly employed throughout embryonic development and dictate the establishment of several cell lineages through mesenchyme-epithelial inductive processes and the interactions of neurons with muscle, glia, and Schwann cells. Likewise, the potent combination of signaling pathways engaged by the heterodimers drives an aggressive phenotype of tumors of secretory epithelia, including breast and lung cancers. This review highlights recent structural insights into the mechanism of ligand-induced heterodimer formation, and concentrates on signaling pathways employed by ErbB-2 and ErbB-3 in normal and in malignant cells. 相似文献
19.
Gianluca Sala Ilario Giovanni Rapposelli Reza Ghasemi Enza Piccolo Sara Traini Emily Capone Cosmo Rossi Angela Pelliccia Annalisa Di Risio Maurizia D'Egidio Nicola Tinari Raffaella Muraro Stefano Iacobelli 《Translational oncology》2013,6(6):676-IN9
ErbB-3 (HER-3) receptor is involved in tumor progression and resistance to therapy. Development of specific inhibitors impairing the activity of ErbB-3 is an attractive tool for cancer therapeutics. MP-RM-1, a murine monoclonal antibody targeting human ErbB-3, has shown anticancer activity in preclinical models. With the aim to provide novel candidates for clinical use, we have successfully generated a humanized version of MP-RM-1. The humanized antibody, named EV20, abrogates both ligand-dependent and ligand-independent receptor signaling of several tumor cell types, strongly promotes ErbB-3 down-regulation, and efficiently and rapidly internalizes into tumor cells. Furthermore, treatment with EV20 significantly inhibits growth of xenografts originating from prostatic, ovarian, and pancreatic cancers as well as melanoma in nude mice. In conclusion, we provide a novel candidate for ErbB-3-targeted cancer therapy. 相似文献
20.
B. J. Appelmelk A. M. J. J Verweij-van Vught H. Brade J. Cohen J. J. Maaskant L. G. Thijs D. M. Mac Laren 《Engineering in Life Science》1990,10(1):13-22
In this paper we review evidence in favor of a protective role for antibodies directed at the lipopolysaccharide (LPS) core region of Gram-negative bacilli. It will be shown that, given the right animal model, polyclonal antisera raised against O-antigen lacking, rough mutant strains, can be shown to protect animals against lethal sepsis due to a variety a bacterial species. Evidence for a protective role obtained from clinical studies will also be discussed. It will be stressed how difficult it is to prove that the antibodies directed against the LPS-core, and not other proteins present in the polyclonal anti-rough strain antisera, were responsible for the observed protective effects. The solution to some of these problems is the production of monoclonal antibodies (Mabs). We will discuss the findings that purified anti-core Mabs may give false-positive outcomes of protection studies, due to the induction of tolerance by small amounts of contaminating endotoxin, present in the antibody preparation. The need to administer the Mab therapeutically instead of prophylactically to the test animals, will be stressed. It will be shown how we succeeded in determining the epitope-specificities of several anti-core antibodies. It will be shown also that use of Elisa alone for characterization of anti-core Mabs may lead to false conclusions concerning epitope specificities. We will prove that synthetic KDO-(=ketodeoxycctanate) and lipid A-containing antigens are indispensable tools for thorough characterization of Mabs. Using this methodology we were able to discriminate between specific antigen-antibody interactions, and the ability of some Mabs to bind “non-specifically” to hydrophobic surfaces. The relationship between epitope specificity and protective effects of anti-core Mabs will be described. Finally, we will demonstrate that a KDO-specific Mabs is capable of protecting mice against lethal sepsis; in other words, it will be shown conclusively that Mabs to defined epitopes in LPS core region are cross-protective. 相似文献