Crystal Structure of Human TWEAK in Complex with the Fab Fragment of a Neutralizing Antibody Reveals Insights into Receptor Binding.

The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases

Expression and localization of claudins-3 and -12 in transformed human brain endothelium

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to characterize the hCMEC/D3 cell line, an <it>in vitro </it>model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12.</p> <p>Findings</p> <p>hCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function <it>in vitro </it>and also recommended for routine hCMEC/D3 culture.</p> <p>Conclusions</p> <p>These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics <it>in vitro</it>.</p

A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.

We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation

Brain Shuttle Neprilysin reduces central Amyloid-β levels.

Reducing Amyloid β (Aβ) in the brain is of fundamental importance for advancing the therapeutics for Alzheimer`s disease. The endogenous metallopeptidase neprilysin (NEP) has been identified as one of the key Aβ-degrading enzymes. Delivery of NEP to the brain by utilizing the Brain Shuttle (BS) transport system offers a promising approach for clearing central Aβ. We fused the extracellular catalytic domain of NEP to an active or inactive BS module. The two BS-NEP constructs were used to investigate the pharmacokinetic/pharmacodynamics relationships in the blood and the cerebrospinal fluid (CSF) in dose-response and multiple dosing. As previously shown, NEP was highly effective at degrading Aβ in blood but not in the CSF compartment after systemic administration. In contrast, the NEP with an active BS module led to a significant CSF exposure of BS-NEP, followed by substantial Aβ reduction in CSF and brain parenchyma. Our data show that a BS module against the transferrin receptor facilitates the transport of an Aβ degrading enzyme across the blood-brain barriers to efficiently reduce Aβ levels in both CSF and brain

Brain Shuttle Antibody for Alzheimer’s Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode

Summary: Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target. : Weber et al. show that a Brain Shuttle antibody against amyloid-β prevents plaque formation mediated by the effector function. Their study reveals that the effector function is hidden in the periphery, due to the unique binding mode of the Brain Shuttle, but becomes fully active in the brain. Keywords: blood-brain barrier, Alzheimer’s disease, antibody effector function, brain delivery, Brain Shuttle, antibody engineerin

128.1 is a high affinity antibody that competes successfully with MEM-189 for binding to TfR in ELISA and in contrast to MEM-189 shows no pH dependence.

<p><b>A</b>: Competition ELISA described in Materials and Methods showing binding of 128.1 to the extracellular subunit of TfR in the presence (▪) or absence (▴) of a pre-block by 5 µg/ml MEM-189. Similarly, binding of MEM-189 in the presence (□) or absence (▵) of 5 µg/mL 128.1. <b>B</b>, Binding ELISA described in Materials and Methods showing binding of the 128.1 anti-human transferrin receptor antibody (▪,□) and MEM-189 antibody (•,○) to the extracellular subunit of the human TfR at pH 7.4 (▪,•) or at pH 5.5 (□,○).</p