We present the complete amino acid sequence of a non-mammalian antithrombin together with a structural comparison to its mammalian homologs. In the majority of cases, but not all, the analyses indicate high degrees of primary structural identity within regions of predicted or demonstrated functional importance. We have also taken advantage of the avian model to define the ontogeny of antithrombin mRNA expression. Northern analyses indicate chicken antithrombin mRNA levels to be detectable in the liver early during development and increase to reach adult levels just prior to hatching. Although no sex-dependent differences in antithrombin mRNA levels were detected, the administration of estrogen to male roosters was observed to cause a 3-fold decrease in antithrombin mRNA levels, suggesting that the hormone may alter circulating antithrombin levels via a pre-translational mechanism.
Critical quality attributes (CQA) are physical, chemical, biological or microbiological properties or characteristics that must be within an appropriate limit, range or distribution to ensure the desired product quality, safety and efficacy. For monoclonal antibody therapeutics that rely on fraction crystalizable (Fc)-mediated effector function for their clinical activity, the terminal sugars of Fc glycans have been shown to be critical for safety or efficacy. Different glycosylation variants have also been shown to influence the pharmacodynamic and pharmacokinetic behavior while other Fc glycan structural elements may be involved in adverse immune reactions. This review focuses on the role of Fc glycans as CQAs. Fc glycan information from the published literature is summarized and evaluated for impact on patient safety, immunogenicity, bioactivity and pharmacodynamics/pharmacokinetics.
AKR (Avian Knotted-Related) was the first example of a vertebrate homeodomain protein with a highly divergent Ile residue at position 50 of the DNA-recognition helix. The protein was cloned from a liver cDNA expression library of a day-9 chick embryo by virtue of its ability to bind to the F' site in the proximal promoter of the avian apoVLDLII gene. Expression of the apoVLDLII gene is completely estrogen dependent, and mutation or deletion of the F' site decreases estrogen inducibility 5- to 10-fold. Subsequent data indicated that AKR is capable of repressing the hormone responsiveness of the apoVLDLII promoter, specifically through binding to F'. Involvement of the F' site in the hormone-dependent activation of apoVLDLII gene expression, as well as AKR-mediated repression, strongly suggests that both positive and negative regulatory factors interact with this site. Although several mammalian proteins have now been isolated whose homeodomains share many of the structural features of AKR, including the Ile at position 50, little is known of their functions in vivo or the identities of the genes they regulate. Consequently, the elements through which they exert their effects and the structural determinants of their binding specificities remain largely uncharacterized. In this study, we defined the sequence specificity of binding by AKR using polymerase chain reaction-assisted optimal site selection and determined the affinity with which the protein binds to both the optimized site and the F' site. Additionally, we generated a three-dimensional model of the AKR homeodomain binding to its optimized site and probed the validity of the model by examining the consequences of mutating amino acid residues in recognition helix 3 and the N-terminal arm on the binding specificity of the homeodomain. Finally, we present evidence that the F' site itself may act as an estrogen response element (ERE) when in the vicinity of imperfect or canonical EREs and that AKR can repress hormone inducibility mediated via this site.
<div>Abstract<p>Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRα ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRα was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRα-mediated signaling results in significant inhibition of tumor growth <i>in vivo</i>. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRα in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.</p></div>
The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity.
A validated bioassay is used to measure the potency of commercial lots, and as such, must be accurate, precise, and fit for its intended purpose. Regulatory expectations for a bioassay include a characterization of features, such as accuracy, precision, linearity, and range. The journey of a bioassay typically starts in a development lab, where it is initially qualified and used to support the release and stability testing of clinical lots. As a program moves through the different clinical phases, it may be optimized further, used to support process development, or transferred to new laboratories, with each activity generating additional bioassay data. Finally, the bioassay is fully validated as part of the transfer to the commercial quality control testing laboratories. In this work, rather than capturing the data from each study as a separate, independent report, it is proposed that, beginning with the qualification study, the accuracy and precision of the bioassay be continuously characterized, with each subsequent study result building upon the preceding ones. We call this approach continuous qualification. Such a proposition is naturally carried out using Bayesian statistical methods in which the historical study data is used to construct prior knowledge that is blended with the current study data. By doing so, the bioassay accuracy and precision may be assessed with high confidence well ahead of commercial manufacturing. Further, by following the total-variance approach, the method also allows for a robust construction of system suitability and control limits for potency.
Regulatory bodies worldwide consider N-glycosylation to be a critical quality attribute for immunoglobulin G (IgG) and IgG-like therapeutics. This consideration is due to the importance of posttranslational modifications in determining the efficacy, safety, and pharmacokinetic properties of biologics. Given its critical role in protein therapeutic production, we review N-glycosylation beginning with an overview of the myriad interactions of N-glycans with other biological factors. We examine the mechanism and drivers for N-glycosylation during biotherapeutic production and the several competing factors that impact glycan formation, including the abundance of precursor nucleotide sugars, transporters, glycosidases, glycosyltransferases, and process conditions. We explore the role of these factors with a focus on the analytical approaches used to characterize glycosylation and associated processes, followed by the current state of advanced glycosylation modeling techniques. This combination of disciplines allows for a deeper understanding of N-glycosylation and will lead to more rational glycan control.
<div>Abstract<p>Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRα ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRα was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRα-mediated signaling results in significant inhibition of tumor growth <i>in vivo</i>. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRα in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.</p></div>
