&amp;lsquo;Epitope Fingerprinting&amp;rsquo; Using Overlapping 20-mer Peptides of the MUC1 Tandem Repeat Sequence

Schol, Dick J.; Meulenbroek, M.F.A.; Snijdewint, F.G.M.; vonMensdorff-Pouilly, Silvia; Verstraeten, Rob A.; Murakami, Fumihiro; Kenemans, P.; Hilgers, J.

doi:10.1159/000056503

Cited by 31 publications

(23 citation statements)

References 14 publications

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“…The majority of MUC1 monoclonal antibodies (MAbs) [35][36][37] are directed to the PDTRP sequence on the repeat domain. This immunodominant region attains its native conformation on synthetic peptides with more than two repeats [38].…”

Section: Discussionmentioning

confidence: 99%

“…The higher inhibition caused by the modified 60-mer peptides, especially in the case of the IgM-positive samples, may be due to diminished steric hindrance among the individual antibody molecules because of a wider separation of the epitopes. Preliminary results obtained on 20 mer overlapping peptides suggest that, unlike mouse MAbs which are generally directed to the PDTR region of the tandem repeat sequence [37], human MUC1 antibodies are also frequently directed to epitopes in the STAPPAHGV region of the MUC1 peptide core [unpubl. obs.].…”

Section: Discussionmentioning

confidence: 99%

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An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

S¹,

Mm²,

Kenemans³

et al. 1998

Tumor Biol

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Introduction: About one-third of breast and ovarian carcinoma patients have circulating antibodies reactive with polymorphic epithelial mucin (MUC1), either free or bound to immune complexes. While the presence of these immune complexes has prognostic significance in breast cancer patients, the significance of free MUC1 antibodies is less clear. The objective of this study was to develop a reliable assay for the accurate determination of circulating free antibodies to MUC1. Material and Methods: We developed an enzyme-linked immunosorbent assay (ELISA) (PEM.CIg) employing a 60 mer peptide (a triple tandem repeat sequence of the MUC1 peptide core) conjugated to bovine serum albumin and peroxidase-labeled antihuman immunoglobulin G or M antibodies. The assay was standardized and its analytical performance evaluated. A total of 492 serum samples were obtained from 40 healthy men, from 201 healthy women (including 55 women without a history of pregnancy and 45 pregnant women), and (before primary treatment) from 62 benign breast tumor patients and 190 breast cancer patients. MUC1 serum levels were determined with commercial CA 15-3 tests. Results: Circulating antibodies to MUC1 are present both in healthy subjects and in breast cancer patients. The within- and between-assay coefficients of variation were, respectively, 2 and 12% for the IgG determinations and 1.2 and 3% for the IgM determinations. Correlation coefficients for serially diluted serum samples ranged from 0.9998 to 0.9920 for IgG and from 0.9996 to 0.9818 for IgM determinations. The reactivity of serum samples was partially blocked by the addition of various MUC1 peptides and by MUC1 mucin. The inhibiting effect of modified 60 mer peptides suggests the presence of antibodies directed to more than one epitope. Conclusions: The PEM. CIg assay is a reliable ELISA for measuring free MUC1 antibodies in serum. We are in the process of relating the results obtained in the breast cancer group to disease outcome to evaluate its prognostic significance. In addition, the assay may become a useful tool for vaccine therapy monitoring.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

S¹,

Mm²,

Kenemans³

et al. 1998

Tumor Biol

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“…Numerous MAbs against MUC1 have been generated by immunizing BALB/c mice with various preparations, such as human milk fat globule membranes [1,5], breast cancers or their cell lines [9,13,14], partially purified membrane mucin preparations [9] and also nonglycosylated MUC1 peptides based on tandem repeat sequences [9,10,13]. However, no antibodies have been generated yet against synthetic glycosylated MUC1 peptides.…”

Section: Discussionmentioning

confidence: 99%

“…The mice were immunized 3 times at weekly intervals. Three weeks after the third dose, a fourth was administered, 4 days prior to a fusion with SP2/0 mouse myeloma cells, as described previously [13,14]. All doses were administered intraperitoneally at 100 Ìg/dose.…”

Section: Generation Of Mab Vu-2-g7mentioning

confidence: 99%

Characterization of a New MUC1 Monoclonal Antibody (VU-2-G7) Directed to the Glycosylated PDTR Sequence of MUC1

et al. 2000

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A monoclonal antibody (MAb), VU-2-G7, was generated against a synthetic 60-mer MUC1 triple tandem repeat peptide with N-acetyl-galactosamine (GalNAc) O-linked to the threonine in the PDTR region of each repeat (3M GalNAc). VU-2-G7 and 8 MUC1 MAbs (VU-3-C6, VU-4-H5, 139H2, A76-A/C7, VU-12-E1, BCP9, MF11 and BW835) were tested against various glycosylated and nonglycosylated MUC1 tandem repeat peptides. VU-2-G7 showed strong reactivity with its immunogen, 3M GalNAc, and much lower reactivity with the nonglycosylated 60-mer MUC1 triple tandem repeat peptide. VU-2-G7 showed no reactivity with a 60-mer MUC1 triple tandem repeat peptide modified at the PDTR region or with a 60-mer MUC1 triple tandem repeat peptide with 3 GalNAc per repeat outside the PDTR region (9M GalNAc). In ELISA and flow cytometry, VU-2-G7 ubiquitously reacted with 4 MUC1-expressing breast cancer and 2 ovarian cancer cell lines and with a MUC1-gene-transfected Chinese hamster ovary cell line. The reactivity of VU-2-G7 was always higher than that of VU-4-H5, raised against a nonglycosylated 60-mer MUC1 triple tandem repeat peptide. Immunohistochemical staining of paraffin sections of breast and ovarian tumor tissues showed strong binding of VU-2-G7 predominantly at the cell membrane. The dominant epitope of VU-2-G7 is in the glycosylated PDTR motif of the MUC1 tandem repeat, and this epitope is abundantly present on the surface of tumor cell lines and breast and ovarian tumor tissues. Given the ubiquitously aberrant glycosylation of MUC1 in malignant cells, the production of MAbs against highly purified glycosylated MUC1 tandem repeat peptides may yield MAbs better suited for the immunotherapy of carcinomas than those available at the moment.

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“…37 All the MUC1 peptides recognised by the MoAbs in the transfected DCs appear either to maintain the RPAP sequence at the N terminus allowing binding of MoAbs 436 and Ma552 or to contain other antibody anchor amino acids (P residue for binding of MoAb BC2). This hypothesis is also in agreement with the requirements described for peptide transport in the E.R.…”

Section: Ma552 G V T S a P D T R P A Pmentioning

confidence: 99%

Transfected human dendritic cells to induce antitumor immunity

et al. 2000

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Dendritic cells are professional antigen-presenting cells able to prime naive T lymphocytes and regulate steadily the delicate balance between tolerance and activation during the immune response. In past years several reports have shown that genetically engineered dendritic cells (DCs) can be a powerful tool for inducing an antigen-specific immune response. The use of such modified antigen-presenting cells is a real working hypothesis in preclinical studies and in clinical vaccination approaches for cancer treatment. The definition of optimal transfection conditions for preserving DC survival and functionality is necessary to design a correct immunotherapeutic protocol. Different lipid-based transfec-

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‘Epitope Fingerprinting’ Using Overlapping 20-mer Peptides of the MUC1 Tandem Repeat Sequence

Cited by 31 publications

References 14 publications

An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

Characterization of a New MUC1 Monoclonal Antibody (VU-2-G7) Directed to the Glycosylated PDTR Sequence of MUC1

Transfected human dendritic cells to induce antitumor immunity

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