A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy.

Although recent methods for the engineering of antibody-drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines

Homogeneous Bispecifics by Disulfide Bridging

We report on a chemical platform to generate site-specific, homogeneous, antibody-antibody conjugates by targeting and bridging disulfide bonds. A bispecific antibody construct was produced in good yield through simple reduction and bridging of antibody fragment disulfide bonds, using a readily synthesized bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics

Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging

The advent of Adcetris™ and Kadcyla™, two recently FDA-approved antibody-drug conjugates (ADCs), in the clinic has had a major impact on the treatment of lymphoma and breast cancer patients, respectively, worldwide. Despite these successes many new ADCs fail at various stages of development, often due to shortcomings in the methods used for their assembly. To address this problem we have developed next generation maleimides (NGMs), which specifically re-bridge reduced interchain disulfide bonds and allow the efficient conjugation of small molecules to antibodies, without the need for engineering of the target antibody. The method is site-specific and generates near homogeneous products in good yields. Moreover, adjustment of the reaction conditions allows control of the conjugation in terms of stoichiometry (drug-loading) and site selectivity. Using this method we prepared a series of ADCs from trastuzumab and doxorubicin (DOX) with a controlled drug-to-antibody ratio (DAR) of 1, 2, 3 and 4. All of these constructs were fully active by ELISA and had more than 90% of re-bridged disulfide bonds by CE-SDS when compared to clinical grade antibody. Furthermore, digest experiments of the DAR 2 material revealed that almost all of the drug had been targeted to the Fab arms of the antibody. Thus, NGMs offer a flexible and simple platform for the controlled assembly of ADCs from an antibody

The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity

The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5(+) malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5(+)-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T 'on-target, off-tissue' toxicity are required to enable a clinical impact of this approach in solid malignancies

Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation

In this communication we describe a novel acid-cleavable linker strategy for antibody-drug conjugation. Functional disulfide bridging of the single interchain disulfide bond of a trastuzumab Fab fragment yields a homogeneous antibody-drug conjugate bearing a thiomaleamic acid linker. This linker is stable at physiological pH and temperature, but quantitatively cleaves at lysosomal pH to release the drug payload

Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds

Tuning the properties of maleimide reagents holds significant promise in expanding the toolbox of available methods for bioconjugation. Herein we describe aryloxymaleimides which represent 'next generation maleimides' of attenuated reactivity, and demonstrate their ability to enable new methods for protein modification at disulfide bonds

Power grip, pinch grip, manual muscle testing or thenar atrophy - which should be assessed as a motor outcome after carpal tunnel decompression? A systematic review

<p>Abstract</p> <p>Background</p> <p>Objective assessment of motor function is frequently used to evaluate outcome after surgical treatment of carpal tunnel syndrome (CTS). However a range of outcome measures are used and there appears to be no consensus on which measure of motor function effectively captures change. The purpose of this systematic review was to identify the methods used to assess motor function in randomized controlled trials of surgical interventions for CTS. A secondary aim was to evaluate which instruments reflect clinical change and are psychometrically robust.</p> <p>Methods</p> <p>The bibliographic databases Medline, AMED and CINAHL were searched for randomized controlled trials of surgical interventions for CTS. Data on instruments used, methods of assessment and results of tests of motor function was extracted by two independent reviewers.</p> <p>Results</p> <p>Twenty-two studies were retrieved which included performance based assessments of motor function. Nineteen studies assessed power grip dynamometry, fourteen studies used both power and pinch grip dynamometry, eight used manual muscle testing and five assessed the presence or absence of thenar atrophy. Several studies used multiple tests of motor function. Two studies included both power and pinch strength and reported descriptive statistics enabling calculation of effect sizes to compare the relative responsiveness of grip and pinch strength within study samples. The study findings suggest that tip pinch is more responsive than lateral pinch or power grip up to 12 weeks following surgery for CTS.</p> <p>Conclusion</p> <p>Although used most frequently and known to be reliable, power and key pinch dynamometry are not the most valid or responsive tools for assessing motor outcome up to 12 weeks following surgery for CTS. Tip pinch dynamometry more specifically targets the thenar musculature and appears to be more responsive. Manual muscle testing, which in theory is most specific to the thenar musculature, may be more sensitive if assessed using a hand held dynamometer – the Rotterdam Intrinsic Handheld Myometer. However further research is needed to evaluate its reliability and responsiveness and establish the most efficient and psychometrically robust method of evaluating motor function following surgery for CTS.</p

The macro- and megabenthic fauna on the continental shelf of the eastern Amundsen Sea, Antarctica

In 2008 the BIOPEARL II expedition on board of RRS James Clark Ross sailed to the eastern Amundsen Sea Embayment and Pine Island Bay, one of the least studied Antarctic continental shelf regions due to its remoteness and ice cover. A total of 37 Agassiz trawls were deployed at depth transects along the continental and trough slopes. A total of 5,469 specimens, belonging to 32 higher taxonomic groups and more than 270 species, were collected. Species richness per station varied from 1–55. The benthic assemblages were dominated by echinoderms and clearly different to those in the Ross, Scotia and Weddell seas. Here we present the macro- and megafaunal assemblage structure, its species richness and the presence of several undescribed species

Return of naturally sourced Pb to Atlantic surface waters

Anthropogenic emissions completely overwhelmed natural marine lead (Pb) sources during the past century, predominantly due to leaded petrol usage. Here, based on Pb isotope measurements, we reassess the importance of natural and anthropogenic Pb sources to the tropical North Atlantic following the nearly complete global cessation of leaded petrol use. Significant proportions of up to 30-50% of natural Pb, derived from mineral dust, are observed in Atlantic surface waters, reflecting the success of the global effort to reduce anthropogenic Pb emissions. The observation of mineral dust derived Pb in surface waters is governed by the elevated atmospheric mineral dust concentration of the North African dust plume and the dominance of dry deposition for the atmospheric aerosol flux to surface waters. Given these specific regional conditions, emissions from anthropogenic activities will remain the dominant global marine Pb source, even in the absence of leaded petrol combustion

Homogeneous antibody fragment conjugation by disulfide bridging introduces 'spinostics'

A major obstacle to the efficient production of antibody conjugates for therapy and diagnosis is the non-ideal performance of commonly used chemical methods for the attachment of effector-molecules to the antibody of interest. Here we demonstrate that this limitation can be simply addressed using 3,4-substituted maleimides to bridge and thus functionalize disulfide bonds to generate homogeneous antibody conjugates. This one-step conjugation reaction is fast, site-specific, quantitative and generates products with full binding activity, good plasma stability and the desired functional properties. Furthermore, the rigid nature of this modification by disulfide bridging enables the successful detection of antigen with a spin labeled antibody fragment by continuous-wave electron paramagnetic resonance (cw-EPR), which we report here for the first time. Antigen detection is concentration dependent, observable in human blood and allows the discrimination of fragments with different binding affinity. We envisage broad potential for antibody based in-solution diagnostic methods by EPR or 'spinostics'