7 research outputs found
Manipulating Heat Shock Factor-1 in Xenopus Tadpoles: Neuronal Tissues Are Refractory to Exogenous Expression
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The Redox State of Transglutaminase 2 Controls Arterial Remodeling
While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall
Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition
The 1,3-dipolar cycloaddition of azides with ring-strained
alkynes
is one of the few bioorthogonal reactions suitable for specific biomolecule
labeling in complex biological systems. Nevertheless, azide-independent
labeling of proteins by strained alkynes can occur to a varying extent,
thereby limiting the sensitivity of assays based on strain-promoted
azide–alkyne
cycloaddition (SPAAC). In this study, a subset of three cyclooctynes,
dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]Ânonyne
(BCN), was used to evaluate the azide-independent labeling of proteins
in vitro. For all three cyclooctynes, we show that thiol-yne addition
with reduced peptidylcysteines is responsible for most of the azide-independent
polypeptide labeling. The identity of the reaction product was confirmed
by LC-MS and NMR analysis. Moreover, we show that undesired thiol-yne
reactions can be prevented by alkylating peptidylcysteine thiols with
iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific
azide-dependent labeling is achieved by preincubating proteins containing
reduced cysteines with IAM
A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates
Despite tremendous efforts in the field of targeted cancer therapy with antibody–drug conjugates (ADCs), attrition rates have been high. Historically, the priority in ADC development has been the selection of target, antibody, and toxin, with little focus on the nature of the linker. We show here that a short and polar sulfamide spacer (HydraSpace™, AE Oss, The Netherland) positively impacts ADC properties in various ways: (a) efficiency of conjugation; (b) stability; and (c) therapeutic index. Different ADC formats are explored in terms of drug-to-antibody ratios (DAR2, DAR4) and we describe the generation of a DAR4 ADC by site-specific attachment of a bivalent linker–payload construct to a single conjugation site in the antibody. A head-to-head comparison of HydraSpace™-containing DAR4 ADCs to marketed drugs, derived from the same antibody and toxic payload components, indicated a significant improvement in both the efficacy and safety of several vivo models, corroborated by in-depth pharmacokinetic analysis. Taken together, HydraSpace™ technology based on a polar sulfamide spacer provides significant improvement in manufacturability, stability, and ADC design, and is a powerful platform to enable next-generation ADCs with enhanced therapeutic index
Correction: Verkade, J.M.M.; et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates. Antibodies 2018, 7, 12
The conflict of interest section of the published paper [1] has been updated as follows[...
Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N‑Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody–Drug Conjugates
A robust,
generally applicable, nongenetic technology is presented
to convert monoclonal antibodies into stable and homogeneous ADCs.
Starting from a native (nonengineered) mAb, a chemoenzymatic protocol
allows for the highly controlled attachment of any given payload to
the N-glycan residing at asparagine-297, based on a two-stage process:
first, enzymatic remodeling (trimming and tagging with azide), followed
by ligation of the payload based on copper-free click chemistry. The
technology, termed GlycoConnect, is applicable to any IgG isotype
irrespective of glycosylation profile. Application to trastuzumab
and maytansine, both components of the marketed ADC Kadcyla, demonstrate
a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover,
the superiority of the native glycan as attachment site was demonstrated
by in vivo comparison to a range of trastuzumab-based glycosylation
mutants. A side-by-side comparison of the copper-free click probes
bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed
a surprising difference in conjugation efficiency in favor of BCN,
which could be even further enhanced by introduction of electron-withdrawing
fluoride substitutions onto the azide. The resulting mAb-conjugates
were in all cases found to be highly stable, which in combination
with the demonstrated efficacy warrants ADCs with a superior therapeutic
index
Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition
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