Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24

Abstract

R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy.

Heterogeneity of Monoclonal Antibodies

“…1), and various truncated structures with the loss of the core fucose, GlcNAc, or both. 48,63,[113][114][115][116][117][118][119][120][121][122][123][124][125][126][127] Oligosaccharides of high mannose have also been reported for several antibodies. 33,62,97,103,111,[128][129][130][131] Sialic acid is normally present at a very low level for Fc oligosaccharides.…”

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti‐melanoma mouse IgG3 monoclonal antibody R24

“…Binding studies with gangliosides GD3 and GM1 (reference) were performed on a BIAcore 2000 biosensor (BIAcore, Uppsala, Sweden) equipped with a carboxymethyldextran coated sensor chip CM5 (research grade) according to Catimel et al (1998) and as recently reported in detail by Kemminer et al (2001). The biosensor curves obtained following injection of MAb R24 over immobilized GD3 were subtracted with the control signal obtained with the antibody injected over immobilized GM1 prior to kinetic analysis.…”

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo

“…The ganglioside D3 (GD3) antigen, meanwhile, holds relevance as GD3 expression is found in stressed melanocytes, and likely more prominently so in perilesional skin of vitiligo patients (31). This GD3 antigen is currently targeted for immunotherapy of melanoma as for its expression on membrane and accessibility to antibodies (32,33). Besides, GD3 contributes to melanogenesis, cell growth, and cell dendricity (34,35).…”