GD3 Lactone Research Articles

Randomized multicenter double-blind phase II trial: The immunological adjuvant OPT-821 with or without a trivalent ganglioside vaccine in metastatic sarcoma patients following metastasectomy.

TPS10103 Background: The gangliosides GM2, GD2, and GD3 are strongly expressed in sarcoma and represent novel antitumor targets. We have demonstrated the safety and immunogenicity of individual monovalent ganglioside conjugate vaccines when administered with OPT821, a synthetic saponin adjuvant that augments the T-cell response. Antitumor effects are observed with anti-ganglioside antibodies in preclinical models, with greatest benefit seen in the micrometastatic setting. Patients (pts) with metastatic sarcoma rendered disease-free by surgery are at high risk for disease recurrence, with no therapy demonstrated to improve outcomes. Therefore, this population is ideally suited to test the efficacy of vaccine induced anti-ganglioside antibodies. We thus initiated this randomized double-blind phase II study of OPT821 with or without a trivalent ganglioside vaccine composed of GM2, GD2 lactone and GD3 lactone conjugated to KLH, an immunogenic carrier protein. Methods: A total of 134 pts will be randomized on a 1:1 basis stratified by disease state (relapsed disease vs metastasis at time of diagnosis), disease-free interval and number of relapses. Key inclusion criteria include stage IV sarcoma (locoregional recurrence is not sufficient) rendered disease-free following surgery or multi-modality therapy, no more than 8 weeks since surgery, and no continuous use of anti-inflammatory medications. Pts with Ewings sarcoma, GIST and rhabdomyosarcoma (except pleomorphic/anaplastic) are ineligible. Eligible pts will receive 10 subcutaneous injections in the outpatient setting over 84 weeks. The primary endpoint is progression-free survival (PFS). Secondary objectives include overall survival (OS) and 1- and 3-year PFS rate. Exploratory objectives include correlation of serologic response to outcome, comparison of tumor ganglioside expression at baseline and at recurrence, and analysis of circulating tumor cells. Enrollment began in July 2010. As of January 1, 2012, 83 patients had been recruited from 12 participating sites. Clinicaltrials.gov#: NCT01141491. Funded by Mabvax Therapeutics and NIH (R21CA13386).

Approaches to augmenting the immunogenicity of melanoma gangliosides: from whole melanoma cells to ganglioside-KLH conjugate vaccines.

Gangliosides are neuraminic acid containing glycosphingolipids that are anchored into the lipid bilayer of the plasma membrane by their lipophilic ceramide moiety. They are overexpressed on tissues of neuroectodermal origin, particularly in tumors such as melanomas, sarcomas, neuroblastomas and astrocytomas. With the ganglioside-KLH plus immunological adjuvant QS-21 conjugate vaccine, GM2 and GD2 have been shown to be consistently immunogenic, inducing cytotoxic IgM antibodies in most patients. The immunogenicity of other gangliosides also expressed on melanoma cells such as 9-0-acetyl GD3 and GD3 lactone is currently being tested with this conjugate vaccine approach. From the initiation of our adjuvant vaccine trials in 1975 to the present, the immunogenicity of ganglioside vaccines has increased significantly as vaccine development has progressed. For instance, GM2 antibody responses increased from low titer IgM antibodies induced in occasional patients after whole cell vaccines, to moderate titer IgM antibodies in 86% of patients after GM2/BCG vaccines, to higher titer IgM antibodies in 100% of patients treated with the GM2-KLH plus QS-21 vaccine. These antibodies are capable of mediating complement mediated cytotoxicity of GM2 expressing melanoma cells in the majority of patients and such antibodies, whether naturally produced or vaccine induced, have been associated with a significantly improved disease-free and overall survival. An initial double-blind randomized trial in AJCC Stage III melanoma patients comparing GM2/BCG with BCG alone, demonstrated a 14% improvement in disease-free interval at 4 years and an 11% improvement in overall survival, though neither result achieved statistical significance. Based on these encouraging clinical results and the clearly improved immunogenicity of the GM2-KLH plus QS-21 vaccine compared to the previous GM2/BCG vaccine, the following two large clinical trials are anticipated to begin in 1995-1996. The GM2-KLH plus QS-21 vaccine will be tested in the intergroup setting by ECOG in 450 patients with AJCC Stage II or III melanoma who are free of disease after surgery. Also to be tested in a multicenter trial is a GM2-KLH plus GD2-KLH plus QS-21 vaccine in patients with high risk AJCC Stage II-IV sarcoma after surgical excision of all known disease.

Inhibitory Effects of Milk Gangliosides on the Adhesion ofEscherichia colito Human Intestinal Carcinoma Cells

The effects of milk gangliosides and their derivatives on the adhesion of enterotoxigenic and enteropathogenic Escherichia coli to Caco-2 cells, a human intestinal carcinoma cell line, were investigated. Human milk gangliosides inhibited the adhesion of enterotoxigenic E. coli to Caco-2 cells in the same proportion, regardless of the lactational stage, but bovine milk gangliosides were less effective. The most effective inhibitor was monosialoganglioside 1 (GM1); the adhesion rate of enterotoxigenic E. coli in the presence of GM1 was less than 20% of the positive control. The adhesion of E. coli was also depressed to 31.4% by monosialoganglioside 3 (GM3). However, the inhibitory effect of disialoganglioside 3 (GD3) was less than that of GM3. GD3 lactone, ceramide lactoside, and N-acetylneuraminic acid did not inhibit E. coli adhesion to Caco-2 cells. GM3 also inhibited the adhesion of enteropathogenic E. coli to Caco-2 cells. Thus, these results suggest that GM3 possibly behaves as a physiological component in the intestinal tract of infants to protect them against enteric infections.

Generation of monoclonal antibodies specific for ganglioside lactones: Evidence of the expression of lactone on human melanoma cells

We generated 3 murine monoclonal antibodies (MAbs) specific for ganglioside lactones by immunizing C3H/HeN mice with purified lactones adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. The use of a wide variety of glycolipids, including ganglioside lactones, enabled the precise structures recognized by these MAbs to be elucidated through an ELISA and by immunostaining on thin-layer chromatography. MAb AMR38, which was generated with GM1 lactone, showed restricted specificity, detecting only the GM1 lactone used for immunization. None of the other ganglioside lactones, intact gangliosides (including GM1) or neutral glycolipids tested were recognized. In contrast, MAbs AMR40 and AMR19, which were generated with GD1a lactone and GD3 lactone, respectively, showed broader specificities, recognizing several ganglioside lactones. However, the precise epitopes were different. MAb AMR40 reacted intensely with ganglioside lactones having an external NeuAc alpha 2-->3Gal-sequence (GD1a, GM3, GM1b, GT1b, and IV3NeuAc alpha-nLc4Cer), but not with those having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence. On the other hand, MAb AMR19 reacted with ganglioside lactones having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence (GD3, O-Ac-GD3, GD2, GDlb, GTlb, GQlb and GTla), but not with those having a NeuAc alpha 2-->3Gal- sequence. None of the intact gangliosides or neutral glycolipids tested were recognized by the MAbs. We also determined the expression of ganglioside lactones on human melanoma cells grown in athymic nude mice by means of an immunofluorescence technique.

Ganglioside expression on human malignant melanoma assessed by quantitative immune thin‐layer chromatography

The ganglioside composition of 20 human malignant melanomas and 5 normal tissues (muscle, spleen, kidney, liver and brain) was analyzed by high-performance thin-layer chromatography (HPTLC) and immune HPTLC using a panel of antiganglioside monoclonal antibodies, and quantified by photodensitometry. The most prominent gangliosides were GM3 and GD3, present in all 20 melanomas; however these were expressed in the 5 normal tissues as well. GD2, GM2, GT3 and 9-O-Ac-GD3 were each expressed in at least 17 of 20 melanomas, but distribution on the normal tissues examined was largely restricted to brain. The detection of several additional glycolipids was studied. GMI was highly expressed in normal brain tissue, but was not detected in any melanoma biopsies, and SGPG was detected in neither. Fuc-GMI was identified in 3 melanoma specimens and a base-sensitive ganglioside, not previously identified in melanoma, was detected in 4 of 20 melanomas with the anti-GD2 MAb 3F8. This compound is most likely O-acetylated GD2. GD3 lactones were identified in 16 of 20 melanoma biopsies, however the proportion that are naturally occurring rather than artifacts of extraction is unclear. The total expression of the more restricted gangliosides (GM2, GD2, GT3 and 9-O-Ac-GD3) in these 20 melanomas ranged between 2.4 and 102.5 micrograms/g, representing 8 x 10(6) to 3 x 10(8) ganglioside molecules per cell. This number of tumor-surface antigens provides the rationale for a polyvalent anti-melanoma vaccine containing GM2, GD2, GT3 and 9-O-Ac-GD3.

Antibody response to immunization with ganglioside GD3 and GD3 congeners (lactones, amide and gangliosidol) in patients with malignant melanoma

GD3 is the ganglioside most abundantly expressed on the cell surface of human melanoma, and treatment with a murine MAb recognizing GD3 has induced major responses in a small proportion of patients with melanoma. We have therefore attempted to induce production of GD3 antibodies in melanoma patients by active immunization. We found, however, that vaccination with GD3-expressing melanoma cells or purified GD3 does not result in antibody production. We describe here attempts to overcome the poor immunogenicity of GD3 in patients with melanoma by chemical modification. GD3 lactones, GD3 amide and GD3 gangliosidol were synthesized, and the humoral immune response to these derivatives was analyzed. Immunization of melanoma patients with these GD3 derivatives resulted in production of IgM antibodies and, in the case of GD3 amide, also of IgG antibodies. The antibodies to the GD3 derivatives did not cross-react with GD3. This is in contrast to observations in the mouse, where GD3 lactone I induced antibodies that showed cross-reactivity with GD3. Thus, the human immune response was specifically directed toward the modified epitope, rather than to the native structure.

Antibody Response to Immunization with Purified GD3 Ganglioside and GD3 Derivatives (Lactones, Amide and Gangliosidol) in the Mouse

GD3 is the ganglioside most abundantly expressed on the cell surface of human melanoma, and treatment with a monoclonal antibody recognizing GD3 has induced major responses in a small proportion of patients. However, we have been unable to induce production of GD3 antibodies in melanoma patients by active immunization with GD3-expressing melanoma cells or purified GD3. In this report we describe attempts to increase the immunogenicity of GD3 in the mouse by chemical modification. GD3 lactone I and II, GD3 amide and GD3 gangliosidol were synthesized, and the humoral immune response to these derivatives was compared with the response to unmodified GD3. The GD3 derivatives were more immunogenic than GD3. At a low dose all congeners induced an IgM response, with antibody titers higher than those elicited by low-dose GD3. The gangliosidol and amide derivatives also induced an IgG response. IgM antibodies induced by immunization with GD3 lactone I crossreacted with purified GD3 and GD3-expressing melanoma cells. Titers of GD3 cross-reactive antibodies were slightly higher than after immunization with GD3 itself at the same low dose. IgM and IgG antibodies induced by the other congeners did not cross-react with GD3.

Ganglioside GD3 lactones: polar head group-mediated control of the intermolecular organization

The individual properties and intermolecular organization of ganglioside GD3 and of two of its lactone forms (GD3Lactone I and GD3Lactone II) were studied in lipid monolayers. The formation of the first lactone ring in GD3Lactone I eliminates one negative charge and leads to a decrease of the molecular area at all surface pressures. The intermolecular dispersion energy and collapse pressure are higher in GD3Lactone I compared to those in the parent GD3. The surface potential per unit of molecular surface density and the resultant molecular dipole moment are increased in GD3Lactone I with respect to those in GD3 at comparable values of molecular area. In GD3Lactone I the molecular parameters suggest an oligosaccharide chain oriented similarly to that of GD3. On the average, this is perpendicular to the surface, and the resultant polar head-group dipole moment points away from the interface. In GD3Lactone II the negative charges are eliminated, resulting in considerably larger molecular areas than for GD3 and GD3Lactone I at all pressures. The intermolecular dispersion energy of GD3Lactone II is also greatly diminished and the collapse pressure is further increased compared to those of GD3Lactone I. However, the surface potential per unit molecular surface density and the resultant molecular dipole moment of GD3Lactone II are higher than in GD3 Lactone I at similar values of molecular areas. This is probably due to a positive polar head-group dipole moment contribution induced by the additional lactone ring in GD3Lactone II. These changes result from a distorted conformation of the oligosaccharide chain owing to the presence of fused carbohydrate rings which require a greater intermolecular spacing compared to GD3 and GD3Lactone I.

Strategies for characterization of ganglioside inner esters II—gas chromatography/mass spectrometry

Two novel gas chromatography/mass spectrometry (GC/MS) strategies have been developed to assist in characterization of ganglioside inner esters. In the first, lactonized gangliosides which have been ammonolyzed and permethylated (see previous paper) are subjected to methanolysis and acetylation. This produces partially methylated NeuAc derivatives in which previous involvement of the carboxyl group in an ester linkage is indicated by conversion to an N1, N1-dimethylamide. These can be analyzed by GC/MS using conditions analogous to those used normally in NeuAc methylation analysis, providing glycosidic and ester linkage information in the same procedure. In the second procedure, internally esterified NeuAc hydroxyl groups are located by protection, under neutral conditions, of all free hydroxyl groups of the ganglioside lactone with methoxyethoxymethyl groups, followed by permethylation, methanolysis, and GC/MS analysis of the resulting partially methylated NeuAc methyl ester methyl glycosides as their per-O-trimethylsilylates or per-O-acetates. This procedure has been used to show unambiguously that in GD3 lactones it is the 9-hydroxyl group of the internal NeuAc residue which is esterified by the terminal NeuAc carboxyl. Both of these strategies should have general application in characterizing lactones occurring in gangliosides, NeuAc homo- and heteropolymers, and oligosaccharides or glycopeptides terminated by polysialosyl chains.

High Resolution Proton NMR Studies of Gangliosides: Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.