Physicochemical and biological characterization of 1E10 Anti-Idiotype vaccine

<p>Abstract</p> <p>Background</p> <p>1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)₃, in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained <it>in vivo </it>from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the <it>in vivo </it>to a bioreactor-based method.</p> <p>Results</p> <p>Here, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between <it>in vivo</it>-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models.</p> <p>Conclusions</p> <p>Changes in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice.</p

Glycan labeling strategies and their use in identification and quantification

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed

HIV glycomics and glycoproteomics

The HIV-1 surface glycoprotein, gp120, is made of a rapidly mutating protein core, encoded by the viral genome, and an extensive carbohydrate shield which is synthesized by the host cell. HIV gp120 is a highly glycosylated protein, with an average of 25 potential N-linked glycosylation sites (PNGS). Determination of the site occupancy, microheterogeneity, and chemical structure of glycans attached to the potential glycosylation sites on gp120 have been performed on recombinant gp120 and gp140 by site analysis of glycosylation involving a combination of chromatography and mass spectrometry techniques. These studies were complemented by lectin-binding studies, and finally by mass spectrometric glycosylation analysis of gp120 isolated directly from infectious virions produced in peripheral blood mononuclear cells (PBMCs). In contrast to host cell glycoproteins, gp120 was shown to contain a population of incompletely processed oligomannose-type glycans that interact with host lectins, promote HIV infection, and alter cell signaling. These glycans also form the basis of the epitopes of several highly potent HIV broadly neutralizing antibodies isolated from HIV-infected individuals, making them a key feature for immunogen design. Furthermore, an elevated level of oligomannose-type glycans was evidenced on gp120 isolated from HIV-1 virions produced in PBMCs, compared to recombinant material, along with a subset of highly processed and sialylated, bi-, tri-, and tetra-antennary complex-type glycans. The effect of variation in viral production systems has also been reported, with envelope glycoprotein derived from pseudoviral particles produced in human embryonic kidney (HEK) 293T cells exhibiting predominantly an oligomannose population, compared to gp120 isolated from a single-plasmid infectious molecular clone. The gp120 glycan profile is remarkably similar across primary viral isolates from Africa, Asia, and Europe and consequently represents an attractive target for vaccine development. Finally, glycan remodeling and mutagenesis can also be employed for pseudoviral particle production and recombinant protein expression, to probe broadly neutralizing antibody specificity, structural analysis, and immunogen design.</p