The Breast Tumor-associated Epitope Defined by Monoclonal Antibody 3E1.2 Is an O-linked Mucin Carbohydrate Containing N-Glycolylneuraminic Acid

Medical Innovations Limited, Labrador, Queensland, Australia.
Cancer Research (Impact Factor: 9.33). 12/1991; 51(21):5826-36.
Source: PubMed


The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5-acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins.

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    • "N-Glycolylneuraminic acid (Neu5Gc) is a sialic acid which is synthesized by animals, but not by humans or birds because of the lack of the enzyme CMP-Neu5Ac hydroxylase which converts Neu5Ac to Neu5Gc [1]. However, it can be incorporated in human cells after entering the body [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]. According to Bardor et al. [11], incorporation of Neu5Gc involves a pinocytic/endocytic procedure and the action of lysosomal sialidase as well as lysosomal sialic acid transporter. "
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    ABSTRACT: Background. N-Glycolylneuraminic acid (Neu5Gc) is a sialic acid synthesized by animals, but not by humans or birds. However, it can be incorporated in human cells and can trigger immune response. In the present study, we detected anti-Neu5Gc antibodies in samples of the general population and of patients suffering from hypothyroidism/Hashimoto’s disease, which is known to have autoimmune origin. Methods. Antibodies were measured using enzyme-immunosorbent techniques. Results. Serum anti-Neu5Gc IgG antibodies were higher in patients with hypothyroidism (mean: 14.8 ± 15.9 μg/mL, median: 10.0 μg/mL, p = 0.0003, Mann-Whitney) and even higher in the group with Hashimoto’s thyroiditis (mean: 31.1 ± 16.3 μg/mL, median: 27.2 μg/mL, p = 0.0000, Mann-Whitney) compared to the general population (mean: 5.3 ± 4.7 μg/mL, median : 4 μg/mL). All anti-TPO positive samples had anti-Neu5Gc antibody concentration higher than the mean value of the general population while anti-TPO concentration was increased as anti-Neu5Gc concentration increased. Low concentrations of IgA and IgM antibodies were measured in both general population and patient groups. Conclusion.The increased values of anti-Neu5Gc antibodies in patients with hypothyroidism/Hashimoto’s disease and the correlation of anti-TPO incidence with increased anti-Neu5Gc concentration raise the possibility of an association between anti-Neu5Gc antibody development and autoimmune hypothyroidism.
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    • "A very interesting case is the one of N-glycolylated (NeuGc) gangliosides, since these glycolipids are not naturally expressed in humans due to a genetic deletion in the gene that codes the CMP-N-acetyl hydroxylase, enzyme that catalyzes the conversion of N-acetyl to N-glycolyl sialic acid (Irie et al., 1998; Irie and Suzuki, 1998; Olson and Varki, 2003). However, both direct and indirect studies have indicated that NeuGc is overexpressed in several human tumors (Devine et al., 1991; Kawai et al., 1991; Marquina et al., 1996; Malykh et al., 2001). The most accepted theory for this phenomenon is the incorporation of NeuGc from dietary sources. "
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    ABSTRACT: Surgery, chemotherapy, and radiation therapy are standard modalities for cancer treatment, but the effectiveness of these treatments has reached a plateau. Thus, other strategies are being explored to combine with the current treatment paradigms in order to reach better clinical results. One of these approaches is the active immunotherapy based on the induction of anti-tumor responses by anti-idiotypic vaccination. This approach arose from Jerne's idiotypic network theory, which postulates that B lymphocytes forms a functional network, with a role in the establishment of the immune repertoires, in the regulation of natural antibody production and even in the establishment of natural tolerance. Due to the large potential diversity of the immunoglobulin variable regions, the idiotypes repertoire can mimic the universe of self and foreign epitopes, even those of non-protein nature, like gangliosides. Gangliosides are sialic acid-containing glycolipids that have been considered attractive targets for cancer immunotherapy, based on the qualitative and quantitative changes they suffer during malignant transformation and due to their importance for tumor biology. Although any idiotype could be able to mimic any antigen, only those related to antigens involved in functions relevant for organism homeostasis, and that in consequence has been fixed by evolution, would be able not only to mimic, but also to activate the idiotypic cascades related with the nominal antigen. The present review updates the results, failures and hopes, obtained with ganglioside mimicking anti-idiotypic antibodies and presents evidences of the existence of a natural response against gangliosides, suggesting that these glycolipids could be idiotypically relevant antigens.
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    • "The lack of expression of this type of sialic acid in humans is due to the inactivating mutation in the CMP-Neu5Ac hydroxylase gene, enzyme responsible for Neu5Gc biosynthesis of Neu5Gc-containing molecules (Chou et al., 1998; Irie and Suzuki, 1998; Olson and Varki, 2003). However, the presence of NeuGc-neuraminic acid residues has been reported in different human tumors, detected by antibodies and by chemical analysis (Higashi et al., 1984, 1988; Hirabagashi et al., 1987; Miyake et al., 1990; Devine et al., 1991; Marquina et al., 1996; Malykh et al., 2001; van Cruijsen et al., 2009; Scursoni et al., 2010), where they are known to be immunogenic (Nguyen et al., 2005). Additionally, recent experimental data suggest that NeuGcGM3 ganglioside is relevant for tumor progression (de León et al., 2006). "
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    ABSTRACT: Neu-glycolyl (NeuGc)-containing gangliosides are attractive targets for immunotherapy with anti-idiotype mAbs, because these glycolipids are not normal components of the cytoplasmic membrane in humans, but their expression has been demonstrated in several human malignant tumors. Racotumomab is an anti-idiotype mAb specific to P3 mAb, an antibody which reacts to NeuGc-containing gangliosides, sulfatides, and other antigens expressed in tumors. Preparations containing racotumomab were able to induce a strong anti-metastatic effect in tumor-bearing mice. Different Phase I clinical trials have been conducted in patients with advanced melanoma, breast cancer, and lung cancer. The results of these clinical trials demonstrated the low toxicity and the high immunogenicity of this vaccine. The induced antibodies recognized and directly killed tumor cells expressing NeuGcGM3. A Phase II/III multicenter, controlled, randomized, double blind clinical trial was conducted to evaluate the effect of aluminum hydroxide-precipitated racotumomab vaccine in overall survival in patients with advanced non-small cell lung cancer. The clinical results of this study showed a significant clinical benefit in the patients who were treated with the anti-idiotype vaccine.
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