The human epithelial carcinoma antigen recognized by monoclonal antibody AE3 is expressed on a sulfoglycolipid in addition to neoplastic mucins

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000 kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray analyses of one of the monoclonal antibodies, AE3, that has been regarded the ‘most carcinoma specific’ in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewis^a/b, Lewis^x/y and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galβ1-3GalNAcβ1-4(3-O-sulfate)Galβ1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.     

The importance of sialic acid,pH and ion concentration on the interaction of uromodulin and complement factor H

Uromodulin (UMOD) can bind complement factor H (cFH) and inhibit the activation of complement alternative pathway (AP) by enhancing the cofactor activity of cFH on degeneration of C3b. UMOD, an N-glycans-rich glycoprotein, is expressed in thick ascending limb of Henle's loop where the epithelia need to adapt to gradient change of pH and ion concentration. ELISA-based cofactor activity of cFH and erythrocytes haemolytic assay was used to measure the impact of native and de-glycosylated UMOD on the functions of cFH. The binding assay was performed under different pH and ion concentrations, using ELISA. The levels of sialic acid on UMOD, from healthy controls and patients with chronic kidney disease (CKD), were also detected by lectin-ELISA. It was shown that removal of glycans decreased the binding between UMOD and cFH and abolished the ability of enhancing C3b degradation. In acidic condition, the binding became stronger, but it reduced as sodium concentration increased. A significant decrease of α-2,3 sialic acids on UMOD was observed in CKD patients compared with that of healthy individuals. The sialic acids on UMOD, local pH and sodium concentration could impact the binding capacity between UMOD and cFH and thus regulate the activation of complement AP.     

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate

Globo H (Fuc12Gal13GalNAc13Gal14Gal14Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc12Gal13GalNAc13Gal. An isomeric structure with an internal GalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.     

Developmentally regulated O-acetylated sialoglycans in the central nervous system revealed by a new monoclonal antibody 493D4 recognizing a wide range of O-acetylated glycoconjugates

We have previously detected an alkali-labile and developmentally regulated antigen in rat embryonic cerebral cortex, which may be 9-O-acetylsialylated GT3 ganglioside (Hirabayashi Y, Hirota M, Suzuki Y, Matsumoto M, Obata K, Ando S (1989) Neurosci Lett 106:193-98). In this study we established a mouse monoclonal antibody, 493D4, that recognizes 9-O-acetyl GT3 ganglioside, but not non-O-acetyl gangliosides. This antibody also reacted with 9-O-acetyl GD3 to a much lesser extent. By using this antibody, we found that O-acetyl GT3 as well as O-acetyl GD3 were expressed strongly in fetal murine cerebral cortex and decreased to an undetectable level after birth. With the assistance of TLC-immunostaining using 493D4 together with Q-Sepharose column chromatography, O-acetyl gangliosides of bovine brain were purified and the structural analysis showed the presence of O-acetyl GD3, O-acetyl LD1, O-acetyl GD2 and O-acetyl GD1b in the adult brain as extremely minor components. Interestingly, the antibody 493D4 could detect O-acetyl sialoglycoproteins in rat brain tissues. One of the major immunoreactive proteins was shown to be synaptophysin, an integral membrane protein specifically present in synaptic vesicles. This monoclonal antibody was therefore useful for sensitive detection of both O-acetylated gangliosides and glycoproteins with O-acetylated sialic acids. This revised version was published online in November 2006 with corrections to the Cover Date.     

Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7; the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes

Lipo-oligosaccharides (LOS) produced by Neisseria gonorrhoeae are important antigenic and immunogenic components of the outer membrane complex. Previously, we showed that murine monoclonal antibody (mAb) 2C7 did not cross-react with human glycosphingolipids but identified the LOS epitope that is widely expressed in vivo and in vitro (Gulati, S., McQuillen, D. P., Mandrell, R. E., Jani, D. B., and Rice, P. A. (1996) J. Infect. Dis. 174, 1223-1237). In the present study, we analyzed the structure of gonococcal strain WG LOS containing the 2C7 epitope and investigated the structural requirements for expression of the epitope. We determined that the WG LOS components are Hep[1]-elongated forms of 15253 LOS that have a lactose on both Hep[1] and Hep[2] (Yamasaki, R., Kerwood, D. E., Schneider, H., Quinn, K. P., Griffiss, J. M., and Mandrell, R. E. (1994) J. Biol. Chem. 269, 30345-30351). In addition, we found that expression of the 2C7 epitope within the LOS is blocked when the Hep[2]-lactose is elongated. Based on the structural data of these LOS and the results obtained from immunochemical analyses, we conclude the following: 1) mAb 2C7 requires both the 15253 OS minimum structure and the N-linked fatty acids in the lipoidal moiety for expression of the epitope; 2) mAb 2C7 binds to the LOS that elongates the lactose on Hep[1] of the 15253 OS, but not the one on Hep[2]; and 3) the 2C7 epitope is expressed on gonococcal LOS despite the presence of human carbohydrate epitopes such as a lactosamine or its N-acetylgalactosaminylated (globo) form. Our study shows that the conserved epitope defined by mAb 2C7 could potentially be used as a safe site for the development of a vaccine candidate.     

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.     

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.     

Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library

The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.     

Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody.

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.     

Characterization of the antigen recognized by a monoclonal antibody MN9: unique transport pathway to the equatorial segment of sperm head during spermiogenesis

Summary MN9, a monoclonal antibody raised against mouse spermatozoa, specifically recognizes the equatorial segment of sperm head in several mammalian species, including humans. Colloidal gold-immuno-electron microscopy of mouse spermatozoa has shown that the antigen is localized in the space between the outer and inner acrosome membranes and on the acrosome membranes at the equatorial segment. Immunoblotting after electrophoresis of spermatozoa from the cauda epididymidis has identified two immunoreactive bands: 38 kDa and 48 kDa in mouse, and 48 kDa in rat. During spermiogenesis in rat, this antigen is transported to the equatorial segment via a unique pathway, first appearing in some cisternae of the endoplasmic reticulum and in the Golgi apparatus of spermatids at around step 3. The antigen can further be found on the vesicles at thetrans-side of the Golgi apparatus, in the matrix of the head cap, and on the head cap membrane in step-4 to step-7 spermatids. The antigen appears to be concentrated at the equatorial segment during late spermiogenesis. Neither the (pro-)acrosomic granule nor the surrounding membrane are required in this pathway. This pathway can be termed the Golgi-head cap tract.     

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody,M2590, to GM3-ganglioside

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside G_M3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of G_M3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of G_M3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.     

N-acetyl-L-galactosaminuronic acid as an epitope common to the O-polysaccharides of Pseudomonas aeruginosa serotype A and H (Homma) recognized by a protective human monoclonal antibody.

We have established a human--mouse heterohybridoma cell line producing a human monoclonal antibody TS-3G2 (IgG gamma 1, K). This monoclonal antibody specifically bound to O-polysaccharides belonging to plural Pseudomonas aeruginosa Homma serotypes, A and H, in contrast to serotype-specific monoclonal antibody which exclusively bound to strains belonging to a single specific serotype. The binding affinity for serotype A strains was higher than that for serotype H strains. Competitive enzyme immunoassay experiments with O-polysaccharide preparations derived from IID 1001, NCTC 8505 (serotype A) and IID 1009 (serotype H) and their derivatives demonstrated that the N-acetyl-L-galactosaminuronic acid residue in O-polysaccharide was essentially involved in the epitope for TS-3G2. Furthermore, a 6-deoxy-hexosamine residue neighboring the reducing terminal of N-acetyl-L-galactosaminuronic acid residues was also concerned with the epitope to some extent. In the experimental infection model of normal mice, the monoclonal antibody TS-3G2 showed a protective activity against both strains of serotype A and H.     

Inhibition of (+)[3H]SKF 10,047 binding to rat brain membranes by FAB fragments from a monoclonal antibody directed against the opioid receptor

Examination of the binding of (+)[³H]SKF 10,047 to rat brain membranes indicated that at a low concentration most of the binding was to the haloperidol-sensitive binding site. Titration curves exhibited a displacement potency order of haloperidol >(+)SKF 10,047 =1,3-diorthotolyl-guanidine(–)SKF 10,047 phencyclidine analogues. The effect of Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor on the binding of(+)[³H]SKF 10,047 to rat brain membranes was examined. The specificity of this antibody for the opioid receptor has been determined by its ability to inhibit the binding of and opioid peptides to rat brain but not the binding of opioid ligands or nonopioid ligands specific for other receptors. The Fab fragments blocked and displaced specifically bound (+)[³H]SKF 10,047 in a titratable manner. Increasing the incubation time of the membranes with the Fab fragments increased the percent inhibition obtained. The Fab fragments acted as noncompetitive inhibitors of (+)[³H]SKF 10,047 binding. A(+)SKF 10,047 binding site in rat brain appears to share a common structural domain with and opioid receptors.     

Effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the influenza pandemic H1N1 2009 vaccine: a randomized controlled trial

Vaccination with the non-adjuvanted split-virion A/California/7/2009 influenza vaccine (pandemic H1N1 2009 vaccine) began in October 2009 in Japan. The present study was designed to assess the effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the pandemic H1N1 2009 vaccine in healthy adult volunteers. One hundred and seventeen participants aged 22 to 62 were randomly assigned to two study groups. In Group 1 (the priming group), participants were first vaccinated with the seasonal trivalent influenza vaccine followed by two separate one-dose vaccinations of the pandemic H1N1 2009 vaccine, whereas in Group 2 (the non-priming group), the participants were first vaccinated with one dose of the pandemic H1N1 2009 vaccine, followed by simultaneous vaccination of the seasonal trivalent vaccine and the second dose of the pandemic H1N1 2009 vaccine. The participants in Group 2 had a seroprotection rate (SPR) of 79.7% and a seroconversion rate (SCR) of 79.7% in the hemagglutination-inhibition test after the first dose of the pandemic H1N1 2009 vaccine, indicating that the pandemic H1N1 2009 vaccine is sufficiently immunogenic. On the other hand, the participants of Group 1 had a significantly weaker antibody response, with a SPR of 60.8% and a SCR of 58.5%. These results indicate that prior vaccination with the seasonal trivalent influenza vaccine inhibits the antibody response to the pandemic H1N1 2009 vaccine. Therefore, the pandemic H1N1 2009 vaccine should be administered prior to vaccination with the seasonal trivalent influenza vaccine.     

Cross‐reactive antibody response to the pandemic A (H1N1) 2009 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan

The cross‐reactivity of antibody to the swine‐origin pandemic influenza A (H1N1) 2009 virus induced by vaccination with a seasonal trivalent influenza vaccine was studied. Paired sera from a cohort of adult volunteers vaccinated with a trivalent seasonal influenza vaccine every year from 2006 to 2008 were collected each year and tested by hemagglutination inhibition (HI) for antibody against the pandemic influenza A (H1N1) 2009 virus. There was little increase in the geometric mean titer overall; a slight increase was detected in the sera obtained in the 2007–2008 season but not in the other two seasons. The proportion of individuals with HI antibody titers ≥ 1:40 did not change significantly from year to year. These results indicate that cross‐reactivity of the antibodies induced by a trivalent seasonal vaccine to the pandemic influenza A (H1N1) 2009 virus is marginal.     

Binding patterns of 51 monoclonal antibodies to peptide and carbohydrate epitopes of the epithelial mucin (MUC1) on tissue sections of adenolymphomas of the parotid (Warthin's tumours): role of epitope masking by glycans

Warthin's tumours provide a unique opportunity to distinguish and compare monoclonal antibodies (mAbs) to the epithelial mucin, MUC1. In this study, we have applied the range of anti-MUC1 antibodies submitted to the ISOBM TD-4 Workshop for this purpose. mAbs and lectins against MUC1-associated carbohydrate epitopes were also included. Among 39 mAbs to peptide epitopes of MUC1, eight distinct types of staining patterns towards the two epithelial cell layers of Warthin's tumours could be observed. A majority of 27 mAbs reacted preferentially (17) or exclusively (10) with columnar cells, whereas 10 mAbs favoured basal cells (1 of them almost exclusively). The observed staining patterns revealed no correlation with the epitopes. However, after carbohydrate-specific periodate oxidation, 33 of the mAbs stained columnar and basal cells equally well, indicating that epitope masking by glycan side chains was in most cases responsible for the different staining patterns. The results demonstrate the profound impact of glycosylation on immunohistochemistry. Among carbohydrate epitopes, sialyl-TF, sialyl-Le(x), sialyl-dimeric Le(x) and Tn were expressed on both columnar and basal cells (the s-TF3 isomer on columnar cells only). The carcinoma-associated Thomsen-Friedenreich epitope was absent.