26 research outputs found
Pre-clinical characterisation of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer's disease
Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimerβs disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species (βseedsβ) containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical developmen
Functional Interchangeability of Late Domains, Late Domain Cofactors and Ubiquitin in Viral Budding
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed
Group Cognitive Behavior Therapy or Social Skills Training for Individuals With a Recent Onset of Psychosis? Results of a Randomized Controlled Trial
Kinetic and docking studies of the interaction of quinones with the quinone reductase active site.
NAD(P)H/quinone acceptor oxidoreductase type 1 (QR1) protects cells from cytotoxic and neoplastic effects of quinones though two-electron reduction. Kinetic experiments, docking, and binding affinity calculations were performed on a series of structurally varied quinone substrates. A good correlation between calculated and measured binding affinities from kinetic determinations was obtained. The experimental and theoretical studies independently support a model in which quinones (with one to three fused aromatic rings) bind in the QR1 active site utilizing a pi-stacking interaction with the isoalloxazine ring of the FAD cofactor
Site-Specific Conjugation to Native and Engineered Lysines in Human Immunoglobulins by Microbial Transglutaminase
The
use of microbial transglutaminase (MTG) to produce site-specific
antibodyβdrug conjugates (ADCs) has thus far focused on the
transamidation of engineered acyl donor glutamine residues in an antibody
based on the hypothesis that the lower specificity of MTG for acyl
acceptor lysines may result in the transamidation of multiple native
lysine residues, thereby yielding heterogeneous products. We investigated
the utilization of native IgG lysines as acyl acceptor sites for glutamine-based
acyl donor substrates. Of the approximately 80 lysines in multiple
recombinant IgG monoclonal antibodies (mAbs), none were transamidated.
Because recombinant mAbs lack the C-terminal Lys447 due to cleavage
by carboxypeptidase B in the production cell host, we explored whether
blocking the cleavage of Lys447 by the addition of a C-terminal amino
acid could result in transamidation of Lys447 by a variety of acyl
donor substrates. MTG efficiently transamidated Lys447 in the presence
of any nonacidic, nonproline amino acid residue at position 448. Lysine
scanning mutagenesis throughout the antibody further revealed several
transamidation sites in both the heavy- and light-chain constant regions.
Additionally, scanning mutagenesis of the hinge region in a Fabβ²
fragment revealed sites of transamidation that were not reactive in
the context of the full-length mAb. Here, we demonstrate the utility
of single lysine substitutions and the C-terminal Lys447 for engineering
efficient acyl acceptor sites suitable for site-specific conjugation
to a range of glutamine-based acyl donor substrates