BackgroundMonoclonal antibodies (mAb) against GD2 ganglioside have been shown to be effective for the treatment of neuroblastoma. Beneficial actions are, however, associated with generalized pain due to the binding of anti- GD2 mAbs to peripheral nerve fibers followed by complement activation. Neuroblastoma cells that express GD2 also express its O-acetyl derivative, O-acetyl- GD2 ganglioside (OAcGD2). Hence, we investigated the distribution of OAcGD2 in human tissues using mAb 8B6 to study the cross-reactivity of mAb 8B6 with human tissues.Methodology/Principal FindingsThe distribution of OAcGD2 was performed in normal and malignant tissues using an immunoperoxydase technique. Anti-tumor properties of mAb 8B6 were studied in vitro and in vivo in a transplanted tumor model in mice. We found that OAcGD2 is not expressed by peripheral nerve fibers. Furthermore, we demonstrated that mAb 8B6 was very effective in the in vitro and in vivo suppression of the growth of tumor cells. Importantly, mAb 8B6 anti-tumor efficacy was comparable to that of mAb 14G2a specific to GD2.Conclusion/SignificanceDevelopment of therapeutic antibodies specific to OAcGD2 may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of antibodies.

Purpose: We previously generated a mouse monoclonal antibody (mAb) specific for the tumorassociated GD2 ganglioside antigen. Here, we describe the development of a chimeric anti-GD2 mAb for more effective tumor immunotherapy. Experimental Design: We cloned the cDNA encoding the immunoglobulin light and heavy chains of the 60C3 anti-GD2 mAb, and constructed chimeric genes by linking the cDNA fragments of the variable regions of the murine light and heavy chains to cDNA fragments of the human n and g1constant regions, respectively. Results: The resultant chimeric anti-GD2 mAb, c.60C3, showed identical binding affinity and specificity to that of its murine counterpart. Both c.60C3 and 60C3 were rapidly internalized by tumor cells at 37jC. When human serum and human natural killer cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell cytotoxicity, respectively, c.60C3 was more effective in killing GD2-expressing tumor cells. However, c.60C3 was ineffective at inducing cell death by apoptosis, although binding of 60C3 induced apoptotic death in vitro. In an in vivo, GD2-expressing, syngeneic tumor model, i.v. injection of c.60C3, but not of 60C3, significantly suppressed tumor growth in mice (P < 0.0005). Conclusion:Immune effector functions mediated by this antibody and its potentially reduced immunogenicity make chimeric c.60C3 a promising therapeutic agent against neuroectodermic tumors.Gangliosides are sialic acid -bearing glycosphingolipids that are expressed in variable amounts on the surface of all mammalian cells (1). Human neuroectodermal tumors, such as melanoma, glioma, neuroblastoma, and small cell lung carcinoma express large amounts of the GD2 ganglioside (2 -5), which, in contrast, is only expressed at very low levels in the peripheral nervous system (6) and the cerebellum (7). Ganglioside GD2 also seems to be involved in several biological functions (reviewed in ref. 8), such as cell recognition (9), cell matrix attachment (10), and cell growth and cell differentiation (11), suggesting that tumor-associated GD2 gangliosides may play a significant role in the tumorigenic phenotypes of these cells.Several murine and mouse/human chimeric anti-GD2 monoclonal antibodies (mAb) have been generated (12 -18); two of which, the murine 3F8 and the mouse/human chimeric Ch14.18 antibodies, are under evaluation in clinical trials (19 -21), with significant measurable regressions of neuroblastoma and melanoma in several patients (19, 21 -23). Ganglioside GD2 on tumor cell surfaces was shown to be a relevant target antigen for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC; refs. 16,17,24,25). Other mechanisms contributing to the antitumor effect of passive immunotherapy with GD2 tumorspecific mAbs involve the anti-idiotype network (26), and direct apoptotic cell death induction (11,27). These findings suggest the usefulness of anti-GD2 antibodies or their derivatives in the therapy of human tumors of neuroectodermal origin. ...