Insights into the Impact of Heterogeneous Glycosylation on the Pharmacokinetic Behavior of Follistatin-Fc-Based Biotherapeutics.

Follistatin 315 heparan sulfate-binding deficient mutant human IgG4 Fc fusion (FST-ΔHBS-Fc) is a follistatin (FST) based Fc fusion protein currently being developed as a novel therapy for several potential indications, including muscle wasting. Previous assessments of the pharmacokinetics and therapeutic activity of FST-ΔHBS-Fc have shown a close association of the exposure-response relationship. The current work builds upon these initial studies by investigating the glycosylation characteristics of FST-ΔHBS-Fc after recombinant expression and its impact on the pharmacokinetics in mice and Cynomolgus monkeys. The data presented indicate that FST-ΔHBS-Fc is heterogeneously glycosylated at the three putative sites in FST when recombinantly expressed in stably transfected Chinese hamster ovary cells. Such carbohydrate heterogeneity, especially with regards to sialic acid incorporation, directly results in sugar-dependent clearance in both mice and Cynomolgus monkeys. Examination of the pharmacokinetics of FST-ΔHBS-Fc molecules containing variable sialic acid content in asialoglycoprotein receptor 1 (ASPGR-1) knockout mice supports the receptor's role as part of the clearance mechanism of the molecules. Based on the evaluation of several variably sialylated lots of material in pharmacokinetic assessments, we define specifications for average sialic acid incorporation into FST-ΔHBS-Fc that result in limited sugar-mediated clearance. Taken together, these studies highlight the importance of establishing an early understanding of the glycosylation/pharmacokinetic relationships of FST-ΔHBS-Fc, which will provide a basis for future application toward optimal systemic drug delivery and dosing strategies.

Pituitary-specific expression and Pit-1 regulation of the rat growth hormone-releasing hormone receptor gene.

The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation.

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.