Evaluation of the antitumor activity of a ganglioside-based vaccine in an N-glycolyl sialic acid-positive mouse melanoma model

MR GABRI; VI SEGATORI; LL OTERO; DE GOMEZ; DF ALONSO

Denver

Congreso; Annual Meeting 2009 AACR; 2009

Sialic acids are components of the glycocalyx in most normal cells as part of membrane proteins and gangliosides. N-glycolyl sialic acids (NeuGc) are a subset of these molecules synthesized by the enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) which is expressed in all mammals except humans. While NeuGc is expressed in somatic mouse cells, there is nearly no information regarding their expression in mouse cancer tissues. Few reports suggest a null presence of this sugar in murine malignant cells, such as the B16 melanoma. By the contrary, in humans NeuGc is a proposed tumor specific antigen in breast carcinoma and melanoma, being an accepted target for immunotherapy. To develop a NeuGc-positive murine melanoma model, we isolated and amplified the CMAH sequence from normal mouse liver and transfected it into B16 cells (B16-H cell line). Transfection resulted in an occurrence of the CMAH mRNA and expression of NeuGc antigen in tumor cells. We observed an increase of the in vitro proliferation rate and adhesion of B16-H cells, as compared with untransfected B16 cells. B16-H cells were tumorigenic after subcutaneous inoculation in syngeneic C57Bl/6 mice, showing a rapid tumor development in most animals with at least 2 x 104 tumor cells per mouse. Antitumor activity of NeuGcGM3/VSSP vaccine was assessed in the B16-H model. The vaccine was obtained by hydrophobic conjugation of NeuGcGM3 ganglioside with outer membrane proteins from N. Meningitidis to form very small size proteoliposomes (provided by the Center of Molecular Immunology, La Habana, Cuba). Mice were inoculated with 2 x 104 B16 or B16-H cells in the subcutis of the right flank. Immunization with 4 intramuscular doses of 200 ug at 2-week intervals significantly reduced tumor incidence and prolonged survival of mice inoculated with transfected B16-H cells expressing NeuGc. No vaccine effects were obtained in animals bearing control B16 tumors. To test the NeuGc expression profile in B16-H cells we conducted a cell cloning method by endpoint dilution cell culture. This method allowed us to isolate several clonal subpopulations with different NeuGc expression levels which demonstrated the heterogeneous expression of B16-H tumors.Although B16-H model seems to be an effective tool in the evaluation of the antitumor activity of a NeuGcGM3 vaccine, the isolation of clonal cell lines with homogeneous NeuGc expression level will help to understand the role of this molecule as a tumor antigen.