Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen.

ABSTRACT: Spheroids cultured directly from tumours can better reflect in vivo tumour characteristics than two-dimensional monolayer culture or three-dimensional culture of established cell lines. In this study, we generated antibodies by directly immunizing mice with primary-cultured living spheroids from human colorectal cancer. We performed phenotypic screening via recognition of the surface of the spheroids and inhibition of their adhesion to extracellular matrices to identify a monoclonal antibody, clone 5G2. The antibody inhibited cell migration in two-dimensional culture and promoted cell detachment. Western blotting and immunohistochemistry detected the 5G2 signal in many colorectal cancer spheroids, as well as patient tumours, but failed to detect in various cell lines examined. We found that 5G2 recognized the Le(a) and Le(c) on N-glycan, and their major carrier proteins were CEACAM5 and CEACAM6. Pre-incubation of the spheroids with 5G2 impaired translocation of integrin β4 from the lateral membrane to the contact interface between the extracellular matrix when embedded in it. As we successfully obtained a functional antibody, which antigen was glycan structures and lost in cell lines, cancer tissue-originated spheroids can be a useful antigen for generating novel anti-cancer antibodies.

SUBMITTER: Sato Y

PROVIDER: S-EPMC4838943 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Generation of a monoclonal antibody recognizing the CEACAM glycan structure and inhibiting adhesion using cancer tissue-originated spheroid as an antigen

Project description:Spheroids cultured directly from tumours can better reflect in vivo tumour characteristics than two-dimensional monolayer culture or three-dimensional culture of established cell lines. In this study, we generated antibodies by directly immunizing mice with cancer tissue-originated spheroids from colorectal cancer. We performed phenotypic screening via recognition of the surface of the spheroids and inhibition of their adhesion to extracellular matrices to identify a monoclonal antibody, clone 5G2. The antibody inhibited cell migration in two-dimensional culture and promoted cell detachment. Western blotting and immunohistochemistry detected the 5G2 signal in many colorectal cancer spheroids, as well as patient tumours, but the signal was reduced in various cell lines. The expression pattern also changed when spheroids were cultured in two-dimensional conditions. We found that 5G2 recognized the N-glycan structure, including Lea and Lec, and their major carrier proteins were CEACAM5 and CEACAM6. Translocation of integrin 4 from the lateral membrane to the extracellular matrix contact interface in the spheroids was delayed by pre-incubation with 5G2. Thus, cancer tissue-originated spheroids can be a useful antigen for generating novel anti-cancer antibodies as the glycan structure is lost in cell lines but retained in cancer tissue-originated spheroids.

2015-12-30 | GSE76405 | GEO

Carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1, 5 and 6 as biomarkers in pancreatic cancer.

Project description:BackgroundAim of this study was to assess the biological function in tumor progression and metastatic process carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1, 5 and 6 in pancreatic adenocarcinoma (PDAC).Experimental designCEACAM knock down cells were established and assessed in vitro and in a subcutaneous and intraperitoneal mouse xenograft model. Tissue and serum expression of patients with PDAC were assessed by immunohistochemistry (IHC) and by enzyme linked immunosorbent assays.ResultsPresence of lymph node metastasis was correlated with CEACAM 5 and 6 expression (determined by IHC) and tumor recurrence exclusively with CEACAM 6. Patients with CEACAM 5 and 6 expression showed a significantly shortened OS in Kaplan-Meier survival analyses. Elevated CEACAM6 serum values showed a correlation with distant metastasis and. Survival analysis revealed a prolonged OS for patients with low serum CEACAM 1 values. In vitro proliferation and migration capacity was increased in CEACAM knock down PDAC cells, however, mice inoculated with CEACAM knock down cells showed a prolonged overall-survival (OS). The number of spontaneous pulmonary metastasis was increased in the CEACAM knock down group.ConclusionThe effects mediated by CEACAM expression in PDAC are complex, though overexpression is correlated with loco-regional aggressive tumor growth. However, loss of CEACAM can be considered as a part of epithelial-mesenchymal transition and is therefore of rather importance in the process of distant metastasis.

| S-EPMC4237406 | biostudies-literature

Novel mouse monoclonal antibodies specifically recognizing β-(1→3)-D-glucan antigen.

Project description:β-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-β-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-β-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing β-glucans with different lengths of oligo-β-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a β-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-β-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these β-glucan-specific mAbs could be useful in combinatorial antifungal therapy.

| S-EPMC6483564 | biostudies-literature

Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

Project description:BackgroundProstate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species.MethodsPerformance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications.ResultsAll mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging.ConclusionsWith apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc.

| S-EPMC7061361 | biostudies-literature

Extended antibody-framework-to-antigen distance observed exclusively with broad HIV-1-neutralizing antibodies recognizing glycan-dense surfaces.

Project description:Antibody-Framework-to-Antigen Distance (AFAD) - the distance between the body of an antibody and a protein antigen - is an important parameter governing antibody recognition. Here, we quantify AFAD for ~2,000 non-redundant antibody-protein-antigen complexes in the Protein Data Bank. AFADs showed a gaussian distribution with mean of 16.3 Å and standard deviation (σ) of 2.4 Å. Notably, antibody-antigen complexes with extended AFADs (>3σ) were exclusively human immunodeficiency virus-type 1 (HIV-1)-neutralizing antibodies. High correlation (R2 = 0.8110) was observed between AFADs and glycan coverage, as assessed by molecular dynamics simulations of the HIV-1-envelope trimer. Especially long AFADs were observed for antibodies targeting the glycosylated trimer apex, and we tested the impact of introducing an apex-glycan hole (N160K); the cryo-EM structure of the glycan hole-targeting HIV-1-neutralizing antibody 2909 in complex with an N160K-envelope trimer revealed a substantially shorter AFAD. Overall, extended AFADs exclusively recognized densely glycosylated surfaces, with the introduction of a glycan hole enabling closer recognition.

| S-EPMC8578620 | biostudies-literature

Human Natural Antibodies Recognizing Glycan Galβ1-3GlcNAc (LeC).

Project description:The level of human natural antibodies of immunoglobulin M isotype against LeC in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors' blood using LeC-Sepharose (LeC is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with LeC, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10-9 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-LeC antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.

| S-EPMC7554730 | biostudies-literature

Molecularly Imprinted Ligand-Free Nanogels for Recognizing Bee Venom-Originated Phospholipase A2 Enzyme.

Project description:In this study, ligand-free nanogels (LFNGs) as potential antivenom mimics were developed with the aim of preventing hypersensitivity and other side effects following massive bee attacks. For this purpose, poly (ethylene glycol) diacrylate was chosen as a main synthetic biocompatible matrix to prepare the experimental LFNGs. The overall concept uses inverse mini-emulsion polymerization as the main route to deliver nanogel caps with complementary cavities for phospholipase A2 (PLA2) from bee venom, created artificially with the use of molecular imprinting (MI) technologies. The morphology and the hydrodynamic features of the nanogels were confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. The following rebinding experiments evidenced the specificity of molecularly imprinted LFNG for PLA2, with rebinding capacities up to 8-fold higher compared to the reference non-imprinted nanogel, while the in vitro binding assays of PLA2 from commercial bee venom indicated that such synthetic nanogels are able to recognize and retain the targeted PLA2 enzyme. The results were finally collaborated with in vitro cell-viability experiments and resulted in a strong belief that such LFNG may actually be used for future therapies against bee envenomation.

| S-EPMC9571764 | biostudies-literature

Generation of highly selective monoclonal antibodies inhibiting a recalcitrant protease using decoy designs.

Project description:Matrix metalloproteinase-12 (MMP-12), also known as macrophage elastase, is a potent inflammatory mediator and therefore an important pharmacological target. Clinical trial failures of broad-spectrum compound MMP inhibitors suggested that specificity is the key for a successful therapy. To provide the required selectivity, monoclonal antibody (mAb)-based inhibitors are on the rise. However, poor production of active recombinant human MMP-12 catalytic domain (cdMMP-12) presented a technical hurdle for its inhibitory mAb development. We hypothesized that this problem could be solved by designing an expression-optimized cdMMP-12 mutant without structural disruptions at its reaction cleft and surrounding area, and thus isolated active-site inhibitory mAbs could maintain their binding and inhibition functions toward wild-type MMP-12. We combined three advances in the field-PROSS algorithm for cdMMP-12 mutant design, convex paratope antibody library construction, and functional selection for inhibitory mAbs. As a result, isolated Fab inhibitors showed nanomolar affinity and potency toward cdMMP-12 with high selectivity and high proteolytic stability. Particularly, Fab LH11 targeted the reaction cleft of wild-type cdMMP-12 with 75?nM binding KD and 23?nM inhibition IC50 . We expect that our methods can promote the development of mAbs inhibiting important proteases, many of which are recalcitrant to functional production.

| S-EPMC7855242 | biostudies-literature

Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.

Project description:We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

| S-EPMC2750034 | biostudies-literature

Generation of a monoclonal antibody recognizing the heavily glycosylated CD45 protein and its application on identifying circulating tumor cells.

Project description:Here, we provide direct evidence that using recombinant proteins expressed in eukaryotic cells as antigen is a practical way to generate monoclonal antibodies (mAbs) against heavily glycosylated proteins. Heavily glycosylated proteins are typically difficult targets for mAb generation, being limited by unsatisfactory affinity and low specificity. Using the heavily glycosylated CD45 protein as an example, we demonstrate the entire process of expressing the protein in eukaryotic cells and using it as an antigen to generate CD45-targeting mAbs in mice. The mAbs generated showed robust affinity and specificity, which are crucial factors for differentiate circulating tumor cells from white blood cells in human breast cancer patient samples. Only 1 cell fusion and 2 cyclic sub-cloning steps were necessary before mAbs with satisfactory performance were obtained.

| S-EPMC5806877 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data