Lipid-Mediated Targeting with Membrane-Wrapped Nanoparticles in the Presence of Corona Formation.

Membrane-wrapped nanoparticles represent a versatile platform for utilizing specific lipid-receptor interactions, such as siallyllactose-mediated binding of the ganglioside GM3 to Siglec1 (CD169), for targeting purposes. The membrane wrap around the nanoparticles not only serves as a matrix to incorporate GM3 as targeting moiety for antigen-presenting cells but also offers unique opportunities for constructing a biomimetic surface from lipids with potentially protein-repellent properties. We characterize nonspecific protein adsorption (corona formation) to membrane-wrapped nanoparticles with core diameters of approximately 35 and 80 nm and its effect on the GM3-mediated targeting efficacy as a function of surface charge through combined in vitro and in vivo studies. The stability and fate of the membrane wrap around the nanoparticles in a simulated biological fluid and after uptake in CD169-expressing antigen-presenting cells is experimentally tested. Finally, we demonstrate in hock immunization studies in mice that GM3-decorated membrane-wrapped nanoparticles achieve a selective enrichment in the peripheral regions of popliteal lymph nodes that contain high concentrations of CD169-expressing antigen-presenting cells.

Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis.

Recent genetic evidence suggests that aberrant glycosphingolipid metabolism plays an important role in several neuromuscular diseases including hereditary spastic paraplegia, hereditary sensory neuropathy type 1, and non-5q spinal muscular atrophy. Here, we investigated whether altered glycosphingolipid metabolism is a modulator of disease course in amyotrophic lateral sclerosis (ALS). Levels of ceramide, glucosylceramide, galactocerebroside, lactosylceramide, globotriaosylceramide, and the gangliosides GM3 and GM1 were significantly elevated in spinal cords of ALS patients. Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosidase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and ß-galactosidase) mediating glycosphingolipid hydrolysis were also elevated up to threefold. Increased ceramide, glucosylceramide, GM3, and hexosaminidase activity were also found in SOD1(G93A) mice, a familial model of ALS. Inhibition of glucosylceramide synthesis accelerated disease course in SOD1(G93A) mice, whereas infusion of exogenous GM3 significantly slowed the onset of paralysis and increased survival. Our results suggest that glycosphingolipids are likely important participants in pathogenesis of ALS and merit further analysis as potential drug targets.

NGcGM3/VSSP vaccine as treatment for melanoma patients.

Gangliosides are glycosphingolipids that are present in the plasma membranes of vertebrates and are involved in multiple cellular processes. In the Center of Molecular Immunology an NGcGM3 ganglioside based vaccine has been developed and is conceptualized as a targeted therapy in cancer. NGcGM3/VSSP vaccine had been used as treatment of metastatic melanoma patients and had showed to be safe and immunogenic. The treatment improved antitumoral response or maintain the response obtained with previous onco-specific treatment as chemotherapy. The results indicate that the vaccine improved overall survival of metastatic melanoma patients after first line-chemotherapy. The clinical trial ongoing currently will allow corroborating these results.

Antitumor effects of exogenous ganglioside GM3 on bladder cancer in an orthotopic cancer model.

OBJECTIVE: To investigate the therapeutic effects of exogenous gangliosides GM3 on human bladder cancer cell lines and the severe combined immunodeficiency mouse model of orthotopic bladder cancer. MATERIALS AND METHODS: Human bladder cancer cell lines YTS-1, T24, 5637, and KK47 were used in the study. In vitro cytotoxicity of GM3 was assessed using the cell counting kit-8. Cell adhesion was determined using a spreading assay. Phosphorylation of epidermal growth factor receptor was determined by Western blotting. In vivo, the orthotopic bladder cancer model was established using severe combined immunodeficiency mice and GM3 was administered intravesically by way of a transurethral catheter. RESULTS: GM3 inhibited the proliferation of all the bladder cancer cell lines tested. The addition of GM3 decreased cell adhesion and epidermal growth factor-dependent phosphorylation of epidermal growth factor receptor. Direct instillation of GM3 into the bladder of the orthotopic model significantly inhibited tumor growth. CONCLUSION: Our results suggest exogenous GM3 as a potential therapeutic agent for treating bladder cancer.

[Effects of GM3 on proliferation, apoptosis and VEGF expression in human lung adenocarcinoma cell line A549 cells].

OBJECTIVE: To determine the effect of exogenous GM3 on proliferation, apoptosis and VEGF expression in human lung adenocarcinoma cell line A549 cells. METHODS: A549 cells were treated with GM3 at different concentrations for 48 hours. MTT assay was used to detect the cell proliferation and flow cytometry was applied to analyze cell apoptosis. RT-PCR was used to detect the expression level of VEGF mRNA and confocal laser scanning microscopy was applied to observe the localization and fluorescence intensity of VEGF. RESULTS: Comparing with the control, being treated with higher than 10 µmol/L GM3 significantly inhibited A549 cell proliferation (P < 0.05), and the suppressive effect could be enhanced following increasing doses. The IC(50) was 412 µmol/L. Comparing with the control, being treated with higher than 40 µmol/L GM3 significantly promoted the apoptotic rate of A549 cells (P < 0.05). Comparing with the control, being treated with higher than 40 µmol/L GM3 significantly decreased the VEGF mRNA level of A549 cells (P < 0.05), and the fluorescence intensity of VEGF distinctly weakened. CONCLUSIONS: Exogenous ganglioside GM3 can inhibit the proliferation, promote apoptosis, and down-regulate the VEGF expression level in A549 cells. This may be considered as two mechanisms of GM3 for its anti-tumor effect by modulating cell apoptosis and angiogenesis.

Inhibition of tumor-induced myeloid-derived suppressor cell function by a nanoparticulated adjuvant.

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.

Epigenetic reduction in invariant NKT cells following in utero vitamin D deficiency in mice.

Vitamin D status changes with season, but the effect of these changes on immune function is not clear. In this study, we show that in utero vitamin D deficiency in mice results in a significant reduction in invariant NKT (iNKT) cell numbers that could not be corrected by later intervention with vitamin D or 1,25-dihydroxy vitamin D(3) (active form of the vitamin). Furthermore, this was intrinsic to hematopoietic cells, as vitamin D-deficient bone marrow is specifically defective in generating iNKT cells in wild-type recipients. This vitamin D deficiency-induced reduction in iNKT cells is due to increased apoptosis of early iNKT cell precursors in the thymus. Whereas both the vitamin D receptor and vitamin D regulate iNKT cells, the vitamin D receptor is required for both iNKT cell function and number, and vitamin D (the ligand) only controls the number of iNKT cells. Given the importance of proper iNKT cell function in health and disease, this prenatal requirement for vitamin D suggests that in humans, the amount of vitamin D available in the environment during prenatal development may dictate the number of iNKT cells and potential risk of autoimmunity.

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8(+) T cells-mediated protection against melanoma B16 challenge.

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8(+) T cells, but not NK1.1(+) cells, are required in the effector phase of the antitumour immune response. The depletion of CD4(+) T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNgamma when were co-cultured with IFNalpha-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine.

Diet-induced changes in membrane gangliosides in rat intestinal mucosa, plasma and brain.

OBJECTIVES: The objective of this study was to determine if dietary gangliosides induce changes in the ganglioside content of intestinal mucosa, plasma and brain and to identify where GM3 and GD3 are localized in the enterocyte membrane. METHODS: Male 18-day-old Sprague-Dawley rats were fed a semipurified diet containing 20% (w/w) fat. The control diet contained triglyceride, reflecting the fat formulation of an existing infant formula. Two experimental diets were formulated by adding sphingomyelin (1% w/w of total fat) or a ganglioside-enriched lipid (0.1% w/w of total fat) to the control diet fat. The ganglioside fraction of ganglioside-enriched lipid diet contained more than 80% GD3. After 2 weeks of feeding, the total and individual ganglioside and cholesterol content was measured in small intestinal mucosa, plasma and brain. RESULTS: The ganglioside-enriched lipid diet significantly increased total gangliosides in the intestinal mucosa, plasma and brain compared with the control diet. The ganglioside-enriched lipid diet significantly increased the level of GD3 (7.5% w/w) in the intestine compared with control (3.2% w/w) while decreasing the level of GM3, the major ganglioside in the intestine. The ratio of cholesterol to ganglioside in the intestinal mucosa, plasma and brain decreased significantly in rats fed the ganglioside-enriched lipid diet compared with controls. Confocal microscopy showed that GM3 is exclusively localized in the apical membrane of the enterocyte whereas GD3 is primarily localized in the basolateral membrane. CONCLUSIONS: : The authors conclude that dietary ganglioside is absorbed in the small intestine and transported to different membrane sites, altering ganglioside levels in the intestinal mucosa, plasma and brain and thus possibly having the potential to change developing enterocyte function (and possibly that of other cell lines).

Immunotherapy of advanced breast cancer with a heterophilic ganglioside (NeuGcGM3) cancer vaccine.

PURPOSE: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Toxicity of a GM3 cancer vaccine in Macaca fascicularis monkey: a 12-month study.

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.