Secretion of inflammatory mediators by rat mast cells (line RBL-2H3) was earlier shown to be inhibited upon clustering a membrane glycoprotein by monoclonal antibody G63. This glycoprotein, named mast cell functionassociated antigen (MAFA), was also shown to interfere with the coupling cascade of the type 1 Fce receptor upstream to phospholipase Cy1 activation by protein-tyrosine kinases.Here we report that the MAFA is expressed as both a monomer and a homodimer. initiates a cascade of biochemical processes coupling it to the secretory responses of these cells. These processes include (i) activation of receptor-associated protein-tyrosine kinases (2) and phosphatases (3), causing transient tyrosine phosphorylation of several cellular proteins (4), (ii) an increase in phosphatidylinositide hydrolysis resulting from phospholipase Cyl activation, and (iii) a rise in the intracellular concentration of free calcium ions (1, 5). The final response to this stimulus is secretion of granule-stored mediators and the de novo synthesis and secretion of arachidonic acid metabolites and cytokines. Several membrane components different from the known FcsRI subunits have been identified by specific monoclonal antibodies (mAbs) (6-9) on the rat mucosal-type mast cells (line RBL-2H3) and shown to modulate FcsRI-mediated secretory response. G63, a mAb that binds a membrane glycoprotein, named mast cell function-associated antigen (MAFA), was shown to inhibit the FcsRI-induced signaling cascade upstream to phospholipase Cyl activation (i.e., before phosphatidylinositide hydrolysis and the transient rise in intracellular [Ca2+]), the culminating degranulation (9), and secretion of interleukin 6 (M.D.G. and I.P., unpublished work). The mAb G63 inhibitory effect required MAFA clustering and was not due to interference with IgE-FceRI interactions (9). Still, crosslinking of FcsRI-IgE complexes by multivalent antigen also led to coclustering of the MAFA with the aggregated FceRI and to enhancement of its internalization (9, 10). The MAFA has been identified by immunoprecipitation with mAb G63 as a glycoprotein with a molecular mass of 28-40 kDa on reducing SDS/PAGE (9). We have now pursued its structural characterization' to resolve the basis for its inhibition of immunologically stimulated mast cell secretion. MATERIALS AND METHODSImmunoprecipitation and N-Deglycosylation of MAFA. Cell preparation, surface radioiodination, and lysis were done as described (9). The MAFA was immunoprecipitated with mAb G63-coated beads (2-4 mg of IgG per g of agarose or Affi-Gel). Sedimented beads were either resuspended in deglycosylation buffer (see below) or boiled in SDS/PAGE sample buffer for elution and electrophoresis of bound MAFA. For cleavage of the N-linked oligosaccharide side chains, the sedimented beads were resuspended in 50 ,lI of 10 mM Tris-HCl, pH 7.0/0.1% SDS/0.5% 2-mercaptoethanol and boiled for 5 min. After cooling the mixture to room temperature, 1% Triton X-100, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride and N-glycosidas...

Active specific immunotherapy is a promising field in cancer research. N-glycolyl (NGc) gangliosides, and particularly NGcGM3, have received attention as a privileged target for cancer therapy. Many clinical trials have been performed with the anti-NGc-containing gangliosides anti-idiotype monoclonal antibody racotumomab (formerly known as 1E10) and the conjugated NGcGM3/VSSP vaccine for immunotherapy of melanoma, breast, and lung cancer. The present paper examines the role of NGc-gangliosides in tumor biology as well as the available preclinical and clinical data on these vaccine products. A brief discussion on the relevance of prioritization of cancer antigens in vaccine development is also included.

In this study, the immunogenicity and toxicity profile of 1E10, an anti-idiotypic vaccine mimicking the N-glycolyl-GM3 ganglioside, was investigated with an extended vaccination protocol. The year-long vaccination scheme consisted of 6 biweekly intradermal injections (induction phase), followed by 10 monthly boosters (maintenance). Nineteen patients with high-risk (stage III) or metastatic breast cancer were vaccinated with different dose levels of 1E10 (0.5, 1, and 2 mg). The humoral and cellular responses to 1E10 and the targeted ganglioside were assessed at baseline and throughout the treatment. Local skin reactions represented the most common adverse event (National Cancer Institute Toxicity Criteria (NCIC) grades I and II), followed by mild flu-like symptoms lasting for 1 to 2 days. Two patients were removed from the study because of vaccine-related hypersensitivity reactions. A third patient was removed from the study after a transient loss of consciousness with uncertain relation to the vaccine. All patients showed a strong antibody response to the targeted ganglioside. In addition, ganglioside-specific T-cell responses were recorded in 5 of 13 evaluable patients. Vaccination with 1E10 was immunogenic and relatively well tolerated. Because similar results were observed with the 3 tested dose levels, the 0.5-mg dose level was selected for future trials.

A glycoprotein identified on RBL-2H3 cells as capable of inhibiting the secretory response induced by the type I Fcε receptor was named mast-cell-function-associated antigen (MAFA). The amino acid sequence deduced from the cloned full-length cDNA has now shown that the MAFA has marked sequence homology with several members of the C-type (calcium-dependent) animal lectin family. The high conservation of cysteinyl residues suggests an important role for intrachain disulfide bonds in attaining its structure and biological activity. We further show that MAFA clustering by monoclonal antibody G63 also inhibits the de novo synthesis and secretion of interleukin-6 induced by the FcεRI stimulus. Though no ligand has yet been identified for the MAFA, experiments using anti-sense oligonucleotides suggest that this novel lectin may have a role in cell adhesion in addition to its immunomodulatory capacity.

Desmopressin (dDAVP) is a well-known peptide analog of the antidiuretic hormone vasopressin, used to prevent excessive bleeding during surgical procedures. dDAVP increases hemostatic mediators, such as the von Willebrand factor (vWF), recently considered a key element in resistance to metastasis. Studies in mouse models and veterinary trials in dogs with locally-advanced mammary tumors demonstrated that high doses of perioperative dDAVP inhibited lymph node and early blood-borne metastasis and significantly prolonged survival. We conducted a phase II dose-escalation trial in patients with breast cancer, administering a lyophilized formulation of dDAVP by intravenous infusion in saline, 30–60 min before and 24 h after surgical resection. Primary endpoints were safety and tolerability, as well as selection of the best dose for cancer surgery. Secondary endpoints included surgical bleeding, plasma levels of vWF, and circulating tumor cells (CTCs) as measured by quantitative PCR of cytokeratin-19 transcripts. Only 2 of a total of 20 patients experienced reversible adverse events, including hyponatremia (grade 4) and hypersensitivity reaction (grade 2). Reactions were adequately managed by slowing the infusion rate. A reduced intraoperative bleeding was noted with increasing doses of dDAVP. Treatment was associated with higher vWF plasma levels and a postoperative drop in CTC counts. At the highest dose level evaluated (2 μg/kg) dDAVP appeared safe when administered in two slow infusions of 1 μg/kg, before and after surgery. Clinical trials to establish the effectiveness of adjunctive perioperative dDAVP therapy are warranted. This trial is registered on www.clinicaltrials.gov (NCT01606072).

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A novel cancer vaccine was obtained by combining GM3 ganglioside with Neisseria meningitidis outer membrane protein complex to obtain very-small-size proteoliposomes (GM3/VSSP). The authors report the results of a phase 1 study of intramuscular administration of GM3/VSSP/Montanide ISA 51 to patients with metastatic melanoma. Twenty-six patients were included in three dose-level cohorts of 120, 240, and 360 mug. The first five doses (induction phase) were given at 2-week intervals, and the remaining four doses were given monthly. Patients were evaluated for dose-related toxicities and antitumor effects. In addition, serum and peripheral blood mononuclear cells were obtained at baseline and throughout treatment to evaluate humoral and cellular immune responses. One episode of severe hypotension and fever was observed in a patient included at the highest dose level. Other toxicities consisted of local reactions at the site of injection and mild fever and chills. Five doses of GM3/VSSP induced an anti-GM3 IgM response in 44% of patients. Serum reactivity was also observed against melanoma cell lines and tumor biopsies. GM3/VSSP was shown to induce very strong in vitro IFNgamma secretion in all evaluated melanoma patients. Furthermore, in one patient IFNgamma secretion was shown to be GM3-specific. A 62% reduction of a mediastinal mass was documented in one patient (partial response), while a second patient benefited from initial disease stabilization followed by tumor reduction in nonmeasurable soft tissue lesions accompanied by vitiligo.

Racotumomab vaccination has a favorable toxicity profile up to a dose of 0.4 mg, and most patients elicited an immune response. Its activity as immunotherapy for neuroectodermal malignancies will be tested in further clinical trials.