A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Tool inhibitors and assays to interrogate the biology of the TRAF2 and NCK interacting kinase.

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development.

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

At-line bioprocess monitoring by immunoassay with rotationally controlled serial siphoning and integrated supercritical angle fluorescence optics.

In this paper we report a centrifugal microfluidic "lab-on-a-disc" system for at-line monitoring of human immunoglobulin G (hIgG) in a typical bioprocess environment. The novelty of this device is the combination of a heterogeneous sandwich immunoassay on a serial siphon-enabled microfluidic disc with automated sequential reagent delivery and surface-confined supercritical angle fluorescence (SAF)-based detection. The device, which is compact, easy-to-use and inexpensive, enables rapid detection of hIgG from a bioprocess sample. This was achieved with, an injection moulded SAF lens that was functionalized with aminopropyltriethoxysilane (APTES) using plasma enhanced chemical vapour deposition (PECVD) for the immobilization of protein A, and a hybrid integration with a microfluidic disc substrate. Advanced flow control, including the time-sequenced release of on-board liquid reagents, was implemented by serial siphoning with ancillary capillary stops. The concentration of surfactant in each assay reagent was optimized to ensure proper functioning of the siphon-based flow control. The entire automated microfluidic assay process is completed in less than 30 min. The developed prototype system was used to accurately measure industrial bioprocess samples that contained 10 mg mL(-1) of hIgG.

Centrifugo-pneumatic valving utilizing dissolvable films.

In this article we introduce a novel technology that utilizes specialized water dissolvable thin films for valving in centrifugal microfluidic systems. In previous work (William Meathrel and Cathy Moritz, IVD Technologies, 2007), dissolvable films (DFs) have been assembled in laminar flow devices to form efficient sacrificial valves where DFs simply open by direct contact with liquid. Here, we build on the original DF valving scheme to leverage sophisticated, merely rotationally actuated vapour barriers and flow control for enabling comprehensive assay integration with low-complexity instrumentation on "lab-on-a-disc" platforms. The advanced sacrificial valving function is achieved by creating an inverted gas-liquid stack upstream of the DF during priming of the system. At low rotational speeds, a pocket of trapped air prevents a surface-tension stabilized liquid plug from wetting the DF membrane. However, high-speed rotation disrupts the metastable gas/liquid interface to wet the DF and thus opens the valve. By judicious choice of the radial position and geometry of the valve, the burst frequency can be tuned over a wide range of rotational speeds nearly 10 times greater than those attained by common capillary burst valves based on hydrophobic constrictions. The broad range of reproducible burst frequencies of the DF valves bears the potential for full integration and automation of comprehensive, multi-step biochemical assay protocols. In this report we demonstrate DF valving, discuss the biocompatibility of using the films, and show a potential sequential valving system including the on-demand release of on-board stored liquid reagents, fast centrifugal sedimentation and vigorous mixing; thus providing a viable basis for use in lab-on-a-disc platforms for point-of-care diagnostics and other life science applications.

Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells.

Sialic acids are structurally diverse molecules that have important roles in the physiological reactions and characteristics of prokaryotes and eukaryotes. These include the ability to mask epitopes on underlying glycan chains and to repulse negatively charged moieties. Here, we describe the metabolism and immunological relevance of sialic acids and outline how their properties have been exploited by the pharmaceutical industry to enhance the therapeutic properties of proteins such as asparaginase and darbepoetin alpha.