Discovery of a novel pseudo ß-hairpin structure of N-truncated amyloid-ß for use as a vaccine against Alzheimer's disease.

One of the hallmarks of Alzheimer's disease (AD) are deposits of amyloid-beta (Aß) protein in amyloid plaques in the brain. The Aß peptide exists in several forms, including full-length Aß1-42 and Aß1-40 - and the N-truncated species, pyroglutamate Aß3-42 and Aß4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aß species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aß3-14, we identified a novel pseudo ß-hairpin structure in the N-terminal region of Aß and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aß1-14 (N-Truncated Amyloid Peptide AntibodieS; the 'TAPAS' vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid-plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aß, which offers new routes for active and passive immunisation against AD.

A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability.

Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide-thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C-S and C-N linked thio-bioconjugates demonstrated C-S functionalized linkers to be cleavable and C-N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments.

Developing an Antibody-Drug Conjugate Approach to Selective Inhibition of an Extracellular Protein.

Antibody-drug conjugates (ADCs) are a growing class of therapeutics that harness the specificity of antibodies and the cell-killing potency of small-molecule drugs. Beyond cytotoxics, there are few examples of the application of an ADC approach to difficult drug discovery targets. Here, we present the initial development of a non-internalising ADC, with a view to selectively inhibiting an extracellular protein. Employing the wellinvestigated matrix metalloproteinase-9 (MMP-9) as our model, we adapted a broad-spectrum, nonselective MMP inhibitor for conjugation and linked this to a MMP-9-targeting antibody. The resulting ADC fully inhibits MMP-9, and ELISA results suggest antibody targeting can direct a nonselective inhibitor.

Disulfide Modified IgG1: An Investigation of Biophysical Profile and Clinically Relevant Fc Interactions.

Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogeneous ADCs, disulfide modification is considered an effective, low-cost, and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallizable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in the rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g., CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of interchain covalent linkages. It was found that by natively rebridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g., thermal stability/aggregation), the natively rebridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to substandard non Fc-specific attributes (thermal stability). These results advocate native disulfide rebridging as an excellent synthetic strategy for forming homogeneous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.

Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones.

Due to their exquisite cysteine-selectivity, excellent stability, and ability to functionally rebridge disulfide bonds, dibromopyridazinediones are emerging as an exciting new class of bioconjugation reagents, particularly in the field of antibody conjugation. Despite this, relatively little work has been performed on the optimisation of their synthesis and subsequent reaction with immunoglobulins. Herein we present a novel synthetic route towards functionalised dibromopyridazinediones, proceeding via an isolatable dibromopyridazinedione-NHS ester. Reaction of this activated intermediate with a variety of amines produces functional dibromopyridazinediones in good to excellent yields. The disulfide rebridging capacity of these reagents was optimised on the clinically relevant IgG1 trastuzumab, resulting in a general method which allows for the generation of site-selectively modified native trastuzumab with over 90% homogeneity (no disulfide scrambling) without the need for protein engineering or enzymatic conjugation.

A Method for Identifying and Developing Functional Group Tolerant Catalytic Reactions: Application to the Buchwald-Hartwig Amination.

Transition-metal catalysis has revolutionized organic synthesis, but difficulties can often be encountered when applied to highly functionalized molecules, such as pharmaceuticals and their precursors. This results in discovery collections that are enriched in substances possessing less desirable properties (high lipophilicity, low polar surface area). Masking groups are often employed to circumvent this problem, which is in opposition to the inherent ideality of these methods for green chemistry and atom economy. A general screening methodology, related to robustness screening described by Glorius et al., builds a broad understanding of the impact of individual functional groups on the success of a transformation under various conditions and provides a simple framework for identifying new conditions that tolerate challenging functional groups. Application of this approach to profile the conditions for the Buchwald-Hartwig amination and rapidly identify bespoke conditions for challenging substrate classes is described.

Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

Ten years of hemovigilance reports of transfusion-related acute lung injury in the United Kingdom and the impact of preferential use of male donor plasma.

BACKGROUND AND METHODS: From 1996 through 2006, 195 cases were reported as transfusion-related acute lung injury (TRALI) to the Serious Hazards of Transfusion scheme and from 1999 onward classified by probability, using clinical features and HLA and/or HNA typing. From late 2003, the National Blood Service provided 80 to 90 percent of fresh-frozen plasma (FFP) and plasma for platelet (PLT) pools from male donors. RESULTS: Forty-nine percent of reports were highly likely/probable TRALI, and 51 percent possible/unlikely. Of 96 investigations, donor antibodies recognizing recipient antigens were found in 73 cases (65%), with HLA Class I in 25 of those (40%), HLA Class II antibodies in 38 (62%), and granulocyte antibodies in 12 (17%). A review in 2003 revealed that the TRALI risk/component was 6.9 times higher for FFP and 8.2 times higher for PLTs than for red blood cells, and that in donors of implicated FFP/PLTs, white blood cell antibodies were found 3.6 times more often than by chance (p

Serious hazards of transfusion: a decade of hemovigilance in the UK.

The Serious Hazards of Transfusion (SHOT) scheme is a UK-wide, independent, professionally led hemovigilance system focused on learning from adverse events. SHOT was established in 1996 as a confidential reporting system for significant transfusion-related events, building an evidence base to support blood safety policy decisions, clinical guidelines, clinician education, and improvements in transfusion practice. Recommendations are formulated by an independent steering group drawn from medical royal colleges and professional bodies. Ten years after its inception, SHOT has analyzed 2630 transfusion safety events, published 8 annual reports with recommendations, and presented data nationally and internationally. These recommendations have underpinned key initiatives, in particular the UK Department of Health "Better Blood Transfusion" strategy. SHOT has encouraged open reporting of adverse events and "near-misses" in a supportive, learning culture, vigilance in hospital transfusion practice, and evaluation of information technology to support this process. The importance of education and training has been emphasized. Detailed analysis of events has identified weaknesses in the transfusion chain. A collaborative initiative between SHOT, the Chief Medical Officer for England's National Blood Transfusion Committee, and the National Patient Safety Agency aims to reduce ABO-incompatible transfusions by improving bedside practice. Cumulative SHOT data have documented the decline in transfusion-related graft vs host disease after implementation of leucodepletion and have highlighted transfusion-related acute lung injury and bacterial contamination of platelets as important causes of death and morbidity. The UK blood services have developed strategies to reduce these risks. Future SHOT data will evaluate the success of these and other blood safety improvements.

The impact of universal leukodepletion of the blood supply on hemovigilance reports of posttransfusion purpura and transfusion-associated graft-versus-host disease.

BACKGROUND: The pathogenesis of posttransfusion purpura (PTP) and transfusion-associated graft-versus-host disease (TA-GVHD) involves patient exposure to donor platelets (PLTs) and T lymphocytes, respectively, which are removed during blood component leukodepletion (LD). STUDY DESIGN AND METHODS: Reports of PTP and TA-GVHD to the UK hemovigilance scheme Serious Hazards of Transfusion (SHOT) from 1996 to 2005 were compared before and after implementation of universal LD during 1999. RESULTS: There were 45 reports of PTP, with a mean of 10.3 per year before universal LD and 2.3 per year afterward (p < 0.001). All patients had received red cells, but before universal LD, only 1 of 31 (3%) cases had also received PLTs, compared to 8 of 14 (57%) afterward (p < 0.001). Thirty-four cases (76%) had human platelet antigen (HPA)-1a antibodies, whereas 11 had antibodies to other HPA specificities, only 1 of which occurred after LD. Two cases reported before LD also had heparin-dependent PLT antibodies. There were 13 reports of TA-GVHD, all fatal, of which only 2 cases of undiagnosed immunodeficiency met current UK criteria for irradiated components. Eight others had one or more risk factors: B-cell malignancy (6), steroids (1), fresh blood (1), and donor-recipient HLA haplotype share (4). Eleven cases were due to non-LD and 2 to LD components (p < 0.001). No cases have been reported since 2001. In an additional 405 cases, nonirradiated components were transfused in error to high-risk recipients, mainly on fludarabine, but none developed TA-GVHD. CONCLUSIONS: These findings suggest that universal LD has further reduced the already low risk of TA-GVHD in immunocompetent recipients and has altered the profile of PTP cases.