Induction of tumor cell death by anti-GD2 monoclonal antibodies (MoAb): Requirement of antibody Fc and a long residence time (slow koff)

Abstract

13507 Background: Anti-GD2 MoAbs have shown clinical activities against neuroblastoma (NB) and other neuroectodermal tumors. Besides complement mediated (CMC) and antibody-dependent cell mediated cytotoxicities (ADCC), they can directly induce cell death. However, MoAb and their genetic constructs differ substantially in death-inducing efficiency. Although multivalent binding is important for these functions, the roles of association on rate (kon), dissociation off rate (koff), valency and Fc have not been carefully defined. Methods: We tested a panel of purified anti-GD2 MoAbs, MoAb fragment, and single chain variable fragment (scFv) fusion protein. The kinetics of in vitro binding was measured by surface plasmon resonance (SPR) using BIACORE 2000. NB cell lines (LAN1, NMB7, BE(2)C, SKNER, SKNJD and SKNLP) were treated with MoAb in vitro, their survivals were studied by the chromogenic assay and IC50 derived using Sigmaplot. Results: The most efficient cell kill against NB was 3F8 (mIgG3) with an average IC50 of 4.7 ug/ml. In contrast, IC50 was >30 ug/ml for 14.G2a (mIgG2a), ME361 (mIgG2a) and S220–51 (mIgG3). It was >100 ug/ml for 3F8 class-switch variant 3G6 (mIgM), 3F8-F(ab’)2(fragment), and 5F11scFv-streptavidin (tetramer). By SPR, the apparent affinity Kd (equilibrium dissociation constant) of 3F8 [1.1x10-8M] was higher than that of 3F8-F(ab’)2 [3.3x10-8M], 14.G2a [9.0x10- 8M], ME361 [4.6x10-7M], and S220–51 [4.6x10-6 M], but lower than that of decameric IgM 3G6 [6.5x10- 9 M] and tetrameric 5F11scFv-streptavidin [2.6x10-9M]. The affinity advantage of 3F8 over other bivalent MoAbs was primarily due to its slower koff [3.0x10-4 sec-1] when compared to other MoAbs [1.1x10- 3-7.3x10-2 sec-1]. Conclusions: Different structural forms of anti-GD2 MoAb have distinct efficiency in inducing NB cell death as well as binding affinity to GD2. Since these properties can affect CMC and ADCC, they may in turn influence the clinical efficacy of immunotherapy. Our findings suggest the importance of an intact IgG3 molecule (the presence of Fc), as well as a long residence time (slow koff). No significant financial relationships to disclose.