Binding Activities and Antitumor Properties of a New Mouse/Human Chimeric Antibody Specific for GD2 Ganglioside Antigen

Abstract

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. ...

Chimeric Antibody c.8B6 to O-Acetyl-GD2 Mediates the Same Efficient Anti-Neuroblastoma Effects as Therapeutic ch14.18 Antibody to GD2 without Antibody Induced Allodynia

“…We were, however, unable to detect apoptosis after tumor cells treatment with either ch14.18 or c.8B6 mAbs in our experiments. The loss of pro-apoptotic activity upon chimerization of mouse mAb 60C3 specific for GD2 ganglioside was also observed previously [17]. Although the cell death mechanism initiated by the mouse mAb 8B6 have not been yet elucidated, our results suggest that the pro-apoptotic activity of anti-GD2 and anti-OAcGD2 mAbs can not fully explained by their sole antigen recognition activity.…”

“…Chimeric anti-OAcGD2 mAb c.8B6 was constructed by joining the complementary deoxyribonucleic acid for the variable region of the murine antibody 8B6 [11] with the human constant regions of the γ1 heavy chain and the κ light chain. The light and heavy chain expression vectors for chimeric antibody were constructed using the same method as described in a previous report [17]. Appropriate light and heavy expression vectors were co-transfected into Chinese hamster ovary (CHO-S) cells and stable transfectants were isolated.…”

A Monoclonal Antibody to O-Acetyl-GD2 Ganglioside and Not to GD2 Shows Potent Anti-Tumor Activity without Peripheral Nervous System Cross-Reactivity

“…In agreement with this earlier study, we did see a slight cross-reactivity of mAb 14G2a against O AcGD2 in immuno-TLC experiments (data not shown). Scatchard analysis further showed that mAb 8B6 and mAb 14G2a displayed equivalent binding affinities for their respective epitopes that were within the range anti-GD2 antibodies [21], [24], [31], [32]. O AcGD2 expression was confirmed on all of the 12 neuroblastoma tumor sections tested.…”

“…Since in vitro cell culture experiments have shown that various anti-GD2 mAbs inhibit tumor cell growth by directly inducing apoptosis [8], [21], [22], we studied whether the mAb 8B6 displayed the same effects on tumor cell viability. To test for the antitumor activity of mAb 8B6, we choose the EL4 cell line because it is tumorigenic in syngeneic immunocompetent C57Bl/6 mice and because it was used previously in many preclinical studies with anti-GD2 mAbs [23].…”

Determination of Carbohydrate Structure Recognized by Prostate-specific F77 Monoclonal Antibody through Expression Analysis of Glycosyltransferase Genes

“…Among numerous anti-carbohydrate mAbs raised against malignant tumor tissues, several reportedly kill targeted cells through antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis by macrophages (17–20). The mAb F77, which was produced by immunizing mice with the prostate cancer cell line PC-3 (20), is one such cytotoxic antibody.…”