Cellular and humoral immune response to N-glycolyl-GM3

GUTHMANN, MARCELO D; VENIER, CECILIA; LEVY, ESTRELLA MARIEL; CASTRO, MÓNICA A; CINAT, GABRIELA; GÓMEZ, ROBERTO; VÁZQUEZ, ANA MARÍA; FAINBOIM, LEONARDO

Alemania

Congreso; CIMT Cancer Immunotherapy, 8th Annual Meeting,; 2010

The association for Cancer Immunotherapy (CIMT)

Gangliosides are a family of sialylated glycolipids which are normal components of the cell membrane. They have been found to be important actors in multiple aspects of cellular interaction with its environment and with transmembrane signaling. As  such, they are involved in cancer progression and have become the focus of several immunotherapeutic approaches. Not all gangliosides are equally immunogenic. N-acetyl-GM3,   the main gangliosides on the cell surface and the most abundant ganglioside in normal serum, is one of the most immunologically tolerated member of the family. In contrast, N-glycolyl-GM3, is not expressed in human normal tissues due to a species-specific genetic mutation that abrogates the biosynthesis of N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been reported, however, in human tumors, and its presence might be derived from dietary sources or a yet unknown alternate synthesis pathway. Nglycolyl-GM3 is expressed in melanoma, breast  and lung cancer cells, and is highly immunogenic. As a result, it has been considered as a target of choice for immunotherapy.  We investigated with an extended vaccination protocol the immunogenicity and toxicity profile of racotumomab, an anti-idiotypic vaccine mimicking N-glycolyl-GM3. The year-long vaccination scheme consisted of six bi-weekly intradermal injections followed by 10 monthly boosters. Nineteen patients with high risk (stage III) or metastatic breast cancer were vaccinated with different dose levels of racotumomab (0.5, 1 and 2 mg). The humoral and cellular responses to racotumomab and to the targeted ganglioside were assessed at baseline and throughout the treatment. Anti-idiotype antibodies and anti-N-glycolyl-GM3 IgM and IgG antibodies were determined by ELISA. Serum antibody reactivity was also tested against P3X63 Ag8 653 murine myeloma cells and B16 murine melanoma cells. Frequency of N-glycolyl-GM3—reactive cells was calculated by IFNã ELISPOT. For that purpose, cryopreserved PBMC and autologous DCã ELISPOT. For that purpose, cryopreserved PBMC and autologous DC  were incubated with N-glycolyl-GM3—containing liposomes. Equivalent amounts of unloaded liposomes were added to control wells. After a 24-h culture, cells in each well were resuspended and transferred to triplicate wells in an IFNã-precoated ELISPOT plate. Subsequent steps for detection of IFNã-secreting cells followed standard ELISPOT procedures. Patients with no response at baseline and with a posttreatment two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 two-fold increase in the number of spots in N-glycolyl-GM3 stimulated wells (versus unstimulated wells) were considered as responsive. All patients showed a strong antibody response to N-glycolyl GM3. In addition, ganglioside-specific IFNã responses were recorded in 5 of 13 evaluable patients.