Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity

Abstract

Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid -containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4 + T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab ¶) 2 but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complementindependent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosislike phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy.

Lonely killers

“…80,81 In contrast to 14F7, the chimeric version of P3 82 was unable to kill GM3(Neu5Gc)-expressing cells. 66 Originally a germ-line IgM, 83 its lower affinity was the explanation given to this observation. 66 This was later proved with a more reactive mutated variant of chimeric P3.…”

“…66 Originally a germ-line IgM, 83 its lower affinity was the explanation given to this observation. 66 This was later proved with a more reactive mutated variant of chimeric P3. By replacing a glutamate by an arginine residue at the heavy chain variable region (VH) of P3, thus increasing the number of this residue in the heavy chain complementary determining region 3 (H-CDR3), an antibody able to bind more strongly to P3 mAb glycolipidic ligands, without affecting its interaction with its anti-idiotypic mAbs was obtained.…”

“…[58][59][60][61]63 A positive correlation between the development of such antibodies and patient survival was found. 63 Despite the lack of direct cytotoxicity by P3 mAb, 66 the antibodies generated by the anti-idiotypic vaccine were able to directly kill GM3(Neu5Gc)-expressing cells. 91 Furthermore, these antibodies induced a 14F7-like type of cell death, including the formation of the membrane lesions on target cells, even at 4°C.…”

“…Moreover, it was demonstrated that in the RE2 mAb killing mechanism, the LFA-1 integrin plays an important role, 70 and 14F7 mAb binding induces phosphorylation of ezrin. 66 This protein is part of a triad known as ERM (ezrin-radixin-moesin), which regulates the association between membrane proteins and the cytoskeleton and participates in signal transduction pathways. 71 Upon phosphorylation, ezrin links the plasma membrane to actin cytoskeleton, and also interacts with transmembrane proteins.…”

“…Furthermore, with the respective cycloheximide and actinomycin inhibitors, it was demonstrated that the cytotoxic activity of 14F7 mAb does not require de novo protein or mRNA synthesis. 66 For KID3/RAV12, it was demonstrated that sodium influx was necessary for cell killing. 48 No data are available for the anti-Porimin mAb.…”