Fibroblast growth factor receptors (FGFRs) are aberrantly activated through single-nucleotide variants, gene fusions and copy number amplifications in 5–10% of all human cancers, although this frequency increases to 10–30% in urothelial carcinoma and intrahepatic cholangiocarcinoma. We begin this review by highlighting the diversity of FGFR genomic alterations identified in human cancers and the current challenges associated with the development of clinical-grade molecular diagnostic tests to accurately detect these alterations in the tissue and blood of patients. The past decade has seen significant advancements in the development of FGFR-targeted therapies, which include selective, non-selective and covalent small-molecule inhibitors, as well as monoclonal antibodies against the receptors. We describe the expanding landscape of anti-FGFR therapies that are being assessed in early phase and randomised controlled clinical trials, such as erdafitinib and pemigatinib, which are approved by the Food and Drug Administration for the treatment of FGFR3 -mutated urothelial carcinoma and FGFR2 -fusion cholangiocarcinoma, respectively. However, despite initial sensitivity to FGFR inhibition, acquired drug resistance leading to cancer progression develops in most patients. This phenomenon underscores the need to clearly delineate tumour-intrinsic and tumour-extrinsic mechanisms of resistance to facilitate the development of second-generation FGFR inhibitors and novel treatment strategies beyond progression on targeted therapy.

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-toGolgi transport steps became rate-limiting in cells with high secretory activity.Immunoglobulins continue to serve as an important model secretory cargo for investigating biochemical processes of oxidative protein folding and subunit assembly in the ER 2 lumen (1). Although immunoglobulins are indispensable as research tools, their potential as human therapeutics has attracted significant interest in recent years in the manufacture of human IgG at large scale (2, 3). Therapeutic human IgGs are often recombinantly produced in variants of CHO cells that were adapted to propagate in suspension culture format. Mammalian cell hosts are often preferred for biopharmaceutical production not only just to achieve desired co-translational and post-translational modifications (4) but also to exploit the stringent protein quality control mechanisms that only allow the secretion of properly folded and correctly assembled proteins (5, 6).To achieve high recombinant protein expression levels in mammalian cells, various cis-acting exogenous nucleotide elements have been engineered into transgene expression cassettes to enhance transcription efficiency, extend message halflife, and increase translation initiation frequency (7,8). Exogenous nucleotide elements also enabled strategies to increase transgene copy number by gene amplification and to suppress epigenetic silencing (9 -12). Despite the success in boosting protein expression per se through these expression vector engineering approaches, such enhancements did not translate into higher glycoprotein secretion partly because post-translational events such as protein folding/assembly and intracellular vesicular transport steps along the secreto...

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

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A model for predicting the sediment transport capacity of turbulent interrill flow on rough surfaces is developed from 1295 flume experiments with flow depths ranging from 3Ð4 to 43Ð4 mm, flow velocities from 0Ð09 to 0Ð65 m s 1 , Reynolds numbers from 5000 to 26 949, Froude numbers from 0Ð23 to 2Ð93, bed slopes from 2Ð7°to 10°, sediment diameters from 0Ð098 to 1Ð16 mm, volumetric sediment concentrations from 0Ð002 to 0Ð304, roughness concentrations from 0 to 0Ð57, roughness diameters from 1Ð0 to 91Ð3 mm, rainfall intensities from 0 to 159 mm h 1 , flow densities from 1002 to 1501 kg m 3 , and flow kinematic viscosities from 0Ð913 to 2Ð556 ð 10 6 m 2 s 1 . Stones, cylinders and miniature ornamental trees are used as roughness elements. Given the diverse shapes, sizes and concentrations of these elements, the transport model is likely to apply to a wide range of ground surface morphologies.Using dimensional analysis, a total-load transport equation is developed for open-channel flows, and this equation is shown to apply to interrill flows both with and without rainfall. The equation indicates that the dimensionless sediment transport rate is a function of, and therefore can be predicted by, the dimensionless shear stress Â, its critical value  c , the resistance coefficient u/uŁ, the inertial settling velocity of the sediment w i , the roughness concentration C r , and the roughness diameter D r . The model has the formTesting reveals that the model gives good unbiased predictions of in flows with sediment concentrations less than 0Ð20. Flows with higher concentrations appear to be hyperconcentrated and to have sediment transport rates higher than those predicted by the model.