g(m3)-ganglioside and Breast-Neoplasms

This study aimed to investigate the immunogenicity of a cancer vaccine consisting of the NeuGcGM3 ganglioside combined with the outer membrane protein complex of Neisseria meningitides to form very small size particles. The vaccine is administered together with Montanide ISA51, as adjuvant treatment for breast cancer patients. After surgical resection and standard first-line chemo/radiotherapy, breast cancer patients in stage II-III were enrolled in a phase III clinical trial and allocated into 2 strata, according to the number of positive lymph nodes [stratum I (0-3); stratum II (≥4)]. Subsequently, patients were randomly assigned to receive the vaccine or placebo. The treatment consisted of 5 vaccine doses (200 μg) every 2 weeks and thereafter monthly reimmunizations to complete 15 doses. The vaccine was well-tolerated and high titers of immunoglobulin M and immunoglobulin G anti-NeuGcGM3 antibodies were similarly detected in each stratum. Hyperimmune sera were able to specifically recognize and kill the NeuGcGM3-expressing L1210 tumor cell line, and these functional capacities were significantly associated with a better clinical outcome in patients of stratum II. Besides, postimmune sera had the capacity to revert in vitro the immunosuppression induced by NeuGcGM3, as measured by the prevention of CD4 downmodulation on human T lymphocytes. Vaccination had no impact on the frequency of regulatory T cells or circulating NK cells. This study demonstrated, for the first time, the immunogenicity of the NeuGcGM3/VSSP/Montanide ISA 51 vaccine in the adjuvant setting and describes the functionality of induced anti-NeuGcGM3 antibodies as potential surrogate biomarkers of clinical benefit.

Topics: Adjuvants, Immunologic; Antibodies; Apoptosis; Bacterial Outer Membrane Proteins; Biomarkers, Pharmacological; Breast Neoplasms; Cancer Vaccines; Cell Line, Tumor; Female; G(M3) Ganglioside; Humans; Immunity, Humoral; Mannitol; Middle Aged; Neisseria meningitidis; Neoplasm Staging; Oleic Acids; Treatment Outcome

The relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, reasons for considering these molecules as potential targets for cancer immunotherapy and diagnosis. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast tumours. Nevertheless, the lack of a direct evidence of this antigenic display in human breast cancer has kept the subject controversial. For the first time, we described herein the "in vivo" detection of GM3(NeuGc) ganglioside in human breast primary tumours using a radioimmunoscintigraphic technique with 14F7, a highly specific anti-GM3(NeuGc) ganglioside monoclonal antibody, labelled with (99m)Tc. In an open, prospective Phase I/II clinical trial, including women diagnosed in stage II breast cancer, the 14F7 monoclonal antibody accumulation in tumours at doses of 0.3 (n=5), 1 (n=5) and 3 mg (n=4) was evaluated. Noteworthy, the immunoscintigraphic study showed antibody accumulation in 100% of patients' tumours for the 1 mg dose group. In turn, the radioimmunoconjugate injected at doses of 0.3 mg or 3 mg of the antibody, was uptaken by 60 and 33.3% of breast tumours, respectively. "In vivo" immune recognition of GM3(NeuGc) in breast tumours reinforces the value of this peculiar target for cancer immunotherapy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Breast Neoplasms; Carcinoma, Ductal, Breast; Dose-Response Relationship, Drug; Female; G(M3) Ganglioside; Humans; Infusions, Intravenous; Middle Aged; Neoplasm Staging; Prospective Studies; Radioimmunodetection; Technetium

A heterophilic ganglioside cancer vaccine was developed by combining NeuGcGM3 with the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). A phase I clinical trial was performed to determine safety and immunogenicity of this vaccine.. Stage III to IV breast cancer patients received up to 15 (200 micro g) doses of the vaccine by intramuscular injection. The first five doses (induction phase) were given at 2-week intervals, with the remaining treatment (maintenance) administered on a monthly basis.. Twenty-one patients, 11 of whom had metastatic disease, were included. Main toxicities included erythema and induration at the injection site, sometimes associated with mild pain, and low-grade fever (World Health Organization grades 1 and 2). All treated patients who completed the induction phase developed anti-NeuGcGM3 antibody titers between 1:1,280 and 1:164,000 immunoglobulin G (IgG), and 1:640 and 1:164,000 IgM. Noteworthy specific IgA antibodies were induced by vaccination in all stage III patients and in three stage IV patients. Serum antibody levels were higher in the stage III patients, with the larger increases observed after week 32. The antiganglioside IgG subclasses were mainly IgG1 and IgG3. Hyperimmune sera increased complement-mediated cytotoxicity versus P3X63 myeloma cells and a marked IgG differential reactivity against human mammary ductal carcinoma samples.. NeuGcGM3/VSSP/Montanide ISA 51 is an unusual immunogenic ganglioside vaccine and also seems to be safe in this small trial. Immunologic surrogates of activity indicate that this reagent warrants further investigation.

Topics: Adult; Aged; Breast Neoplasms; Cancer Vaccines; Complement System Proteins; Enzyme-Linked Immunosorbent Assay; Female; G(M3) Ganglioside; Humans; Immunoenzyme Techniques; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunohistochemistry; Injections, Intramuscular; Middle Aged; Neisseria meningitidis; Neoplasm Staging; Proteolipids; Remission Induction; Treatment Outcome

A phase I clinical trial was conducted in patients with stage III/IV breast cancer who were treated with the anti-idiotype mAb 1E10 specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with antigens expressed on human melanoma and breast carcinoma cells. Patients were treated with 1 or 2 mg of aluminum hydroxide-precipitated 1E10 mAb every other week for six injections. Two patients at each dose were reimmunized 7-9 months after completing the induction phase. In hyperimmune sera from eight of the nine patients who received at least four doses of anti-Id vaccine preparations, strong specific responses were observed both against 1E10 mAb and NeuGc-GM3 ganglioside (Ab3 Id+Ag+). Nonclassical Ab1' antibodies (Id-Ag+) were also elicited by 1E10 mAb vaccine treatment. There were no differences between the two levels of dose tested in relation to toxicity and immunogenicity. No evidence of serious or unexpected effects was observed.

Topics: Adult; Aged; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Binding Sites, Antibody; Breast Neoplasms; Cancer Vaccines; Female; G(M3) Ganglioside; Humans; Immunotherapy, Active; Middle Aged

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-β receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Topics: Bacteriophage T7; Breast Neoplasms; Doxorubicin; Female; G(M3) Ganglioside; Humans; MCF-7 Cells; Nuclear Proteins; Phosphoproteins; Vascular Endothelial Growth Factor A

Gangliosides altered during the pathological conditions and particularly in cancers. Here, we aimed to profile the gangliosides in breast cancer serum and propose potential biomarkers. LC-FTMS method was first used to identify all the ganglioside species in serum, then LC-MS/MS-MRM method was employed to quantitate the levels of gangliosides in serum from healthy volunteers and patients with benign breast tumor or breast cancer. 49 ganglioside species were determined, including GM1, GM2, GM3, GD1, GD3 and GT1 species. Compared to healthy volunteers, the levels of GM1, GM2, GM3, GD1 and GD3 displayed a rising trend in breast cancer patients. In particular, as the major glycosphingolipid component, GM3 showed excellent diagnostic accuracy in cancer serum (AUC > 0.9). PCA profile of the GM3 species showed clear distinction between normal and cancer serum. What's more, ROC curve proved great diagnostic accuracy of GM3 between cancer and benign serum. In addition, GM3 was discovered as a diagnostic marker to differentiate luminal B subtype from other subtypes. Furthermore, a positive correlation between GM3 and Ki-67 status of patients was identified. In conclusion, our results introduced the alteration patterns of serum gangliosides in breast cancer and suggested serum GM3 as a potential diagnostic biomarker in breast cancer diagnosis and luminal B subtype distinction.

Topics: Adult; Aged; Area Under Curve; Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Chromatography, Liquid; Diagnosis, Differential; Female; G(M3) Ganglioside; Gangliosides; Humans; Ki-67 Antigen; Middle Aged; Neoplasms; Principal Component Analysis; Prognosis; ROC Curve; Tandem Mass Spectrometry

Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal, Humanized; Antigens, Neoplasm; Breast Neoplasms; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; Female; G(M3) Ganglioside; Humans; Hybridomas; Immunotherapy; Mice; Mixed Function Oxygenases; Molecular Targeted Therapy; Mutagenesis, Site-Directed; Protein Engineering; RNA, Small Interfering

GM3 is a ganglioside that has been biochemically identified as dominating the cell surface of several human tumours, but is also found on human normal cells at much lower density. Since GM3 is widely distributed in essentially all types of animal cells, there is a conflict with the concepts of tumour-associated antigen, immunogen, and toxicity. We have designed a GM3-based cancer vaccine for the treatment of human breast and melanoma tumours. Prior to the Phase I clinical trial, we carried out a 12-month dose repeated toxicity study in five male Macaca fascicularis monkeys. Four male monkeys were treated with placebo in a similar way. During the study, no differences were observed between control and treated monkeys related to daily clinical observations (other than local damage) including rectal temperature, blood pressure, respiratory and cardiac rates, weight gain, biochemical and hematological parameters (with the exception of transitory pathological changes), and anti-DNA and anti-nuclear antibodies, although treated monkeys consistently developed both IgM- and IgG-specific anti-GM3 antibodies. Sixty per cent of treated monkeys developed moderate local reactions at the injection site, which disappeared without sequels. We concluded that this GM3 cancer vaccine overcame in monkeys the natural tolerance to GM3 ganglioside evidenced by a strong immune response, while the local reactions elicited-were transitory without apparent important systemic toxicity effects.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Neoplasm; Body Weight; Breast Neoplasms; Cancer Vaccines; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dogs; Drug Evaluation, Preclinical; G(M3) Ganglioside; Hematologic Tests; Immunoglobulin G; Immunoglobulin M; Kidney Function Tests; Liver Function Tests; Macaca fascicularis; Male; Melanoma; Proteolipids; Skin Neoplasms; Toxicity Tests

We investigated the effects of combination therapy with N-(4-hydroxyphenyl)retinamide (4-HPR) and tamoxifen (TAM) on estrogen receptor (ER) negative breast cancer, for which no effective supplementary therapy has been established, using the human breast cancer cell line MDA-MB-231. TAM or 4-HPR alone had little antitumor effect, but the combined use of TAM and 4-HPR had a strong cell growth inhibitory effect. Cell cycle analysis by flow cytometry showed an increased frequency of the G2/M phases in the 4-HPR-TAM combination group. Measurement of 3H-TAM incorporation into the cell showed that, compared with the TAM group, the 4-HPR-TAM combination group incorporated about 1.45 times more TAM into the cell. Thin-layer chromatographic analysis of changes in the cell membrane ganglioside GM3 showed a marked increase in GM3 in the 4-HPR-TAM combination group. We speculate that the administration of TAM in the presence of 4-HPR changes the membrane glycolipid GM3, increasing intracellular TAM concentrations, thus exerting antitumor activity. Presumably, during this process, antitumor effects do not induce cell death but arrest the cell cycle in the G2 phase. Thus, the combined use of TAM and 4-HPR inhibited the growth of the ER-negative breast cancer cell line MDA-MB-231. These results suggest that combination therapy with TAM and 4-HPR can be a potent supplementary therapy also for ER-negative patients in clinical practice.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Chromatography, Thin Layer; Fenretinide; G(M3) Ganglioside; Humans; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured

14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund's adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked immunosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Biomarkers, Tumor; Breast Neoplasms; Cholesterol, VLDL; Female; G(M3) Ganglioside; Glycolipids; Humans; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Organ Specificity

An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Breast Neoplasms; Female; G(M3) Ganglioside; Humans; Mice; Mice, Inbred BALB C; Tumor Cells, Cultured

In vitro immunization of human B-lymphocytes was performed with liposomes containing the monosialoganglioside GM3, with or without either complete tetanus toxoid or a synthetic T helper epitope derived from tetanus toxin (determinant 830-843). The immunized B-cells were Epstein-Barr virus transformed and the human anti-ganglioside antibody response was evaluated using an indirect ELISA against different mono- and disialogangliosides. Clones producing antigen-specific human antibodies of the IgM isotype against the ganglioside GM3 used as the immunogen were selected and one clone, IM-11, was further characterized. In addition, a method of positive selection using GM3-coated magnetic beads has been developed which allowed us to rescue unstable clones. The binding of the human antibody IM-11 to a large panel of glycosphingolipids separated on thin-layer plates was studied. The human MAb IM-11 was found to bind strongly to NeuAcGM3, IV3 NeuAcnLc4 and sulfate containing glycosphingolipids and weakly to NeuGcGM3. Immunohistological staining of melanoma and breast cancer biopsy sections showed a selective reactivity of IM-11 with tumor cells which varied among different tumors.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigens, Neoplasm; B-Lymphocytes; Breast Neoplasms; Carbohydrate Sequence; G(M3) Ganglioside; Humans; Immunization; Melanoma; Molecular Sequence Data

In this study we report the characterization of a human monoclonal antibody (HuMab), L612, that reacts with ganglioside GM3 and has therapeutic application for the treatment of human neoplasms, particularly melanoma. A permanent IgM-secreting Epstein-Barr virus-transformed B-cell line L612 was established. L612 HuMab bound specifically to neoplastic cell lines in culture and in tissue biopsy specimens such as melanoma, colon, breast, and lung cancer. The antibody did not bind to normal cells or biopsy tissue. HuMab L612 showed the highest reactivity to melanoma cells, particularly to those with high concentrations of GM3. Immunostaining on high-performance thin-layer chromatography plates demonstrated that L612 HuMab bound to GM3 purified from melanoma cells. Removal of the sialic acid from GM3 abolished antibody binding. HuMab L612 also reacted to GM4 purified from egg yolk, indicating that it recognizes an NeuAc alpha 2-3 galactose antigen determinant. HuMab L612 heavy and light chains were sequenced and determined to belong to the mu heavy chain variable subgroup III and kappa chain variable subgroup IV families, respectively. The studies indicate that the L612 HuMab has significant therapeutic potential for a wide variety of human cancers.

Topics: Adenocarcinoma; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; B-Lymphocytes; Base Sequence; Breast Neoplasms; Carbohydrate Sequence; Cell Line, Transformed; Cloning, Molecular; Colonic Neoplasms; DNA Primers; Female; G(M3) Ganglioside; Gangliosides; Herpesvirus 4, Human; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Lung Neoplasms; Melanoma; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; Recombinant Proteins; Skin Neoplasms

Ganglioside lactones absent in homologous normal tissues have been found in minute amounts in human gastric and mammary tumours. In mammary gland tumours only the ganglioside GM3 lactone has been identified. Gastric tumours also contain the GM3 lactone; in one case a ganglioside GD3 lactone was identified.

Topics: Breast Neoplasms; G(M3) Ganglioside; Gangliosides; Humans; Lactones; Stomach Neoplasms

Gangliosides of human gastric and mammary tumours and of homologous normal tissues were studied by using biochemical methods and specific antisera. It was found that in most cases GM3, GD3 and GM1 are predominant gangliosides, whereas several polar components are minor ones. A comparison of the relative amount of ganglioside fractions revealed that in gastric tumours the per cent content of polar compounds is higher than in intact tissue; however, the absolute content of all gangliosides is markedly increased. A comparative study of the composition of mammary tumour and normal tissue gangliosides demonstrated two types of changes: i) the absolute content of all gangliosides in tumour tissue was increased and, ii) the increase in the content of total gangliosides was paralleled with the appearance of a new fraction (presumably GM4), the decrease of the GD3 content and the disappearance of polar gangliosides. A possible mechanism of this effect is discussed.

Topics: Breast Neoplasms; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Stomach Neoplasms

The appearance of Hanganutziu and Deicher (HD) antibody in the sera of patients suffering from various diseases, including malignancies of some organs and liver disorders, was investigated by enzyme-linked immunosorbent assay using N-glycolylneuraminyl-lactosylceramide (HD3) and 4-O-acetyl-HD3 as the antigenic molecules. More than 25% of sera from patients suffering from malignancies, cholelithiasis and liver cirrhosis had HD antibody, whereas none of 41 sera from healthy persons had HD antibody. The percentage of HD antibody-positive patients was similar in stages I, II and III of gastric cancer and recurrence cases. Antibody titers of the positive patients in each stage were also not different from those in each other stage. These results indicated that HD antigenic expression on cancerous tissue is not dependent on the cancerous malignancy. The HD antibody level was elevated after surgical removal of cancerous tissues in 5 of 6 patients examined, indicating that tumor growth absorbed the serum antibody. Serum antibody against 4-O-acetyl-HD3 was detected independently of HD3 antibody in some cases; however, in most cases, correlation between the two antibody titers was observed.

Topics: Aged; Antibodies, Heterophile; Antigens, Neoplasm; Breast Neoplasms; Cholelithiasis; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Esophageal Neoplasms; Female; G(M3) Ganglioside; Humans; Liver Cirrhosis; Lymphoma; Male; Middle Aged; Neoplasms; Pancreatic Neoplasms; Stomach Neoplasms