Production and molecular characterization of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24

R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglios ide GD3 expressed by cells of neuroectodermal origin. The anti-tumor activi ty of R24 has been demonstrated in initial phase I and pilot trials in pati ents suffering from metastatic melanoma. The purpose of this study was to i nvestigate the biotechnological production and particularly the glycosylati on of this clinically important antibody. Growth, metabolism, and IgG produ ction of R24 secreting hybridoma cells were analyzed on 1 L bioreactor benc h scale using repeated-batch mode. The amount of 57 mg of pure mab was obta ined from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the h eavy 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 R2 4 was subjected to both enzymatic deglycosylation using PNGase F and chemic al deglycosylation by hydrazinolysis. Released glycans were structurally ch aracterized by high pH anion exchange chromatography with pulsed amperometr ic detection (HPAEC-PAD), matrix assisted laser desorption ionization time- of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-fligh t (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 mug sialic acid per mg protein, which splits into 0.243 mug Neu5Gc and 0.217 mug Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthe rmore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interac tions were measured with an optical biosensor (BIAcore). From the structura l data obtained in this study it is concluded that glycosylation of the ant ibody may be important in the clinical outcome of targeted anti-cancer immu notherapy.