Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.

The specificity of 26 monoclonal antibodies against the CA 125 antigen was investigated in two phases of the ISOBM TD-1 workshop. The binding specificity was studied using CA 125 immunoextracted by specific antibodies immobilized on various solid phases, or on the surface of human cell lines. Immunometric assays using all possible antibody combinations were used to study the topography of antibody binding sites on the antigen. We conclude that the CA 125 antigen carries only two major anti-genic domains, which classifies the antibodies as OC125-like (group A) or Mil-like (group B). One antibody, OV 197, showed binding specificity related to some of the OC125-like antibododies, but was classified into a separate group C. The OC125-like group of antibodies has four subgroups with different binding specificities. These are A1 = OC 125 and K 95, A2 = K 93, A3 = B43.13, and A4 = ZS 33, B27.1 and CCD 247. Binding of nonlabelled OC 125 or K 95 to CA 125 caused a marked increase in binding of labelled OV 197 to the complex. This conformational change was not observed with any other antibody combinations. Antibody B43.13 could form immunometric assay combinations particularly with antibodies of subgroup A4, indicating that the B43.13 epitope is in the periphery of the binding area of OC125-like antibodies. The M11 -like group of antibodies is more homogenous with strong cross-inhibition between most antibodies. Only one antibody, ZR 38, would form an immunoassay combination with other M11 -like antibodies and thus represents a distinct subgroup. The main group of M11-like antibodies are M 11, ZR 45, MA602-6, K 91, OV 185, K 101, K 90, K 96, K 97, K 102, CCD 242, 145-9, and 130-22. Antibody OV 197 binds to a domain designated C and is unique, as stated above. Antibody pairs from any two of the three groups may be used in immunometric assays. Three antibodies were not studied by complete cross-inhibition due to low affinity (OV 198 and K 100) or lack of material (MA602-1). OV 198 and K 100 are most likely OC125-like and MA602-1 is Ml 1-like. Antibody affinity was estimated with labelled antigen in solution or with antigen adsorbed on microtiter wells. Western blot analysis showed staining both in the stacking gel and corresponding to a molecule of 200 kDa. There was a marked difference between the antibodies in their ability to bind to CA 125 immobilized on a membrane. Strongest binding was observed with the M11-like antibodies, particularly M 11, K 96, K 97, MA602-6, 145-9. Antibodies belonging to the subgroup A4 were the only OC 125-like antibodies which reacted well with CA 125 in Western analysis. Digestion of CA 125 with proteolytic enzymes showed it to be particularly sensitive to trypsin cleavage. However, no low molecular weight fragments with preserved immunoreactivity were found.

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for non-invasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal route showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through non-invasive intranasal route can be a strategy for designing low-dose vaccines.

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Escherichia coli O157:H7-specific antibodies (immunoglobulin Y [IgY]) were isolated by the waterdilution method from the egg yolk of chickens that were immunized with E. coli O157:H7 whole cells. The specificbinding activity of IgY against E. coli O157:H7 as determined by the enzyme immuno assay showed high levels of activity against bacterial whole cells. IgY binding activity was further demonstrated to have an inhibitory effect on E. coli O157:H7 growth in a liquid medium. The antibacterial function of IgY appeared to result from the interaction of IgY with surface components of E. coli O157:H7, as proven from observation of immunofluorescence and immunoelectron microscopy.

A novel class of 1-(4-methanesulfonylphenyl and 4-aminosulfonylphenyl)-5-[4-(1-difluoromethyl-1,2-dihydropyrid-2-one)]-3-trifluoromethyl-1H-pyrazole hybrid cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory anti-inflammatory agents was designed. Replacement of the tolyl ring present in celecoxib by the N-difluoromethyl-1,2-dihydropyrid-2-one moiety provided compounds showing dual selective COX-2/5-LOX inhibitory activities. 1-(4-Aminosulfonylphenyl)-5-[4-(1-difluoromethyl-1,2-dihydropyrid-2-one)]-3-trifluoromethyl-1H-pyrazole exhibited good anti-inflammatory (AI) activity (ED(50) = 27.7 mg/kg po) that compares favorably with the reference drugs celecoxib (ED(50) = 10.8 mg/kg po) and ibuprofen (ED(50) = 67.4 mg/kg po). The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel 5-LOX pharmacophore for the design of cyclic hydroxamic mimetics for exploitation in the development of COX-2/5-LOX inhibitory AI drugs.

A chemical selection procedure has been used to prepare a hybrid hybridoma cell line (P4C1) following fusion of two previously established hybridomas secreting antiperoxidase and antisubstance P, respectively. P4C1 secretes bispecific monoclonal antibody alongside the two parental antibodies, with no visible inactive heterologous heavy-light chain pairs. The bispecific monoclonal antibody is thus easy to purify in excellent yields. The advantage of its monovalency for one antigen and simultaneous binding of a marker enzyme has been explored for its potential use in competitive immunoassays. Its use in immunocytochemistry led to major improvements in sensitivity, signal-to-noise ratio, simplification of staining procedures, and ultrastructural preservation of subcellular elements. Particularly remarkable was that, unlike conventional procedures, the immunoreaction with the bispecific monoclonal antibody was homogeneously distributed across the entire thickness of a 50-,um section.Hybrid hybridomas are derived by fusing two (or more) antibody-producing cells. The resulting cells secrete hybrid immunoglobulins exhibiting the binding characteristics of the two individual parental hybridomas in a single molecule (1, 2). These bispecific monoclonal antibodies (bs mAb) are structurally bivalent but functionally univalent for each combining site.The potential of such hybrid molecules has been studied with the antisomatostatin-antiperoxidase hybrid hybridoma P4C7 (1,2). This was derived by fusing spleen cells from immunized rats and a hypoxanthine/aminopterin/thymidinesensitive antiperoxidase rat hybridoma. Although this approach offers several advantages, it is apparent that quality and affinity ofthe derived bs mAb remains a matter of chance and depends on the contribution of the spleen partner. It was desirable to derive hybrid hybridomas derived from two well-characterized hybridomas to compare their performance relative to that of the parental lines. This paper describes the use of a chemical selection procedure to generate a hybrid hybridoma derived from the fusion of a wellcharacterized antisubstance P (anti-SP) hybridoma (NC1/34) (3) ¶ and an antiperoxidase hybridoma (YP4) (1). The derived antiperoxidase-anti-SP bs mAb (P4C1) was found to be better for the histochemical detection of antigenic sites in a singlestep incubation than the mAb NC1/34 developed by peroxidase-antiperoxidase (PAP) methods (4). was conjugated to bovine serum albumin (BSA) using glutaraldehyde (5).Fusion Protocol. Fusion was between hybridomas YP4 (1) [a rat antiperoxidase hybridoma (IgGl)] and NC1/34 anti-SP (a rat-mouse hybrid secreting rat IgG2a). The sensitivity of NC1/34 to emetine-an irreversible inhibitor of protein synthesis-was determined. Typically, 106 cells were incubated with doubling dilutions of the drug for 1 hr at 370C in 10 ml of Dulbecco's modified Eagle's medium (DMEM). The cells were collected by centrifugation, washed, resuspended in 10 ml of DMEM at 370C for 30 min to allow efflux of any residual free drug in ...

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of “colony clusters” from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a β1-integrin isoform with a high degree of N-glycosylation. Treatment with a β1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of β1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated β1-integrin.