Differential influence on antibody dependent cellular phagocytosis by different glycoforms on therapeutic Monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs), particularly of the IgG1 subclass, are capable of effector function activities that may be important for their mechanism of action. One such effector function activity is Antibody Dependent Cellular Phagocytosis (ADCP), which has been shown to be mediated primarily through the activating FcγR, FcγRIIa, on macrophages and neutrophils. The critical quality attributes that are the most impactful and predictive of ADCP activity, and therefore most suitable to monitor during IgG1 antibody manufacturing, are not well established. Primary cell assays for ADCP are often laborious and subject to donor to donor variability, making such assays less desirable for product characterization. By developing and employing an ADCP reporter gene assay, we have been able to determine with high sensitivity the glycan structures that can impact FcγRIIa mediated ADCP across multiple different IgG1 antibodies. Interestingly we observed that some IgG1 antibodies are very potent mediators of ADCP while others do not mediate ADCP even though they possess other effector function activities (ADCC and CDC). Additionally, we find that ADCP by different IgG1 antibodies has markedly different sensitivity to glycan species, with one antibody demonstrating a surprisingly strong influence of ß-galactosylation and high mannose levels.

Glycan engineering reveals interrelated effects of terminal galactose and core fucose on antibody-dependent cell-mediated cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been identified as one of the potentially critical effector functions underlying the clinical efficacy of some therapeutic immunoglobin G1 (IgG1) antibodies. It has been well established that higher levels of afucosylated N-linked glycan structures on the Fc region enhance the IgG binding affinity to the FcγIIIa receptor and lead to increased ADCC activity. However, whether terminal galactosylation of an IgG1 impacts its ADCC activity is less understood. Here, we used a new strategy for glycan enrichment and remodeling to study the impact of terminal galactose on ADCC activity for therapeutic IgG1s. Our results indicate that the degree of influence of terminal galactose on in vitro ADCC activity depends on the presence or absence of the core fucose, which is typically linked to the first N-acetyl glucosamine residue of an N-linked glycosylation core structure. Specifically, terminal galactose on afucosylated IgG1 mAbs enhanced ADCC activity with impact coefficients (ADCC%/Gal%) more than 20, but had minimal influence on ADCC activity on fucosylated structures with impact coefficient in the range of 0.1-0.2. Knowledge gained here can be used to guide product and process development activities for biotherapeutic antibodies that require effector function for efficacy, and also highlight the complexity in modulating the immune response through N-linked glycosylation of antibodies.

Reversal of pentylenetetrazole-altered swimming and neural activity-regulated gene expression in zebrafish larvae by valproic acid and valerian extract.

RATIONALE: Ethnopharmacology has documented hundreds of psychoactive plants awaiting exploitation for drug discovery. A robust and inexpensive in vivo system allowing systematic screening would be critical to exploiting this knowledge. OBJECTIVE: The objective of this study was to establish a cheap and accurate screening method which can be used for testing psychoactive efficacy of complex mixtures of unknown composition, like plant crude extracts. METHODS: We used automated recording of zebrafish larval swimming behavior during light vs. dark periods which we reproducibly altered with an anxiogenic compound, pentylenetetrazole (PTZ). First, we reversed this PTZ-altered swimming by co-treatment with a well-defined synthetic anxiolytic drug, valproic acid (VPA). Next, we aimed at reversing it by adding crude root extracts of Valeriana officinalis (Val) from which VPA was originally derived. Finally, we assessed how expression of neural activity-regulated genes (c-fos, npas4a, and bdnf) known to be upregulated by PTZ treatment was affected in the presence of Val. RESULTS: Both VPA and Val significantly reversed the PTZ-altered swimming behaviors. Noticeably, Val at higher doses was affecting swimming independently of the presence of PTZ. A strong regulation of all three neural-activity genes was observed in Val-treated larvae which fully supported the behavioral results. CONCLUSIONS: We demonstrated in a combined behavioral-molecular approach the strong psychoactivity of a natural extract of unknown composition made from V. officinalis. Our results highlight the efficacy and sensitivity of such an approach, therefore offering a novel in vivo screening system amenable to high-throughput testing of promising ethnobotanical candidates.