New approaches for biomarker stability determination in regulated bioanalysis: trending, bridging and incurred samples.

Aim: Determining the stability of biomarkers continues to present challenges. Disease states, complex matrices and differences between recombinant and endogenous analytes require new approaches to maintain stability and measure it. In this report, we determine stability for two assays using trending and statistical analysis. Methodology & results: Monitoring trends helps identify out of specification measurements and determine whether concerns are due to the stability of the analyte. We also describe challenges presented when measuring arginase activity in human sputum, a complex matrix, for respiratory diseases. We controlled preanalytical protease activity and collection heterogeneity and monitored incurred sample stability to improve stability of arginine. Conclusion: These new approaches to achieving and determining biomarker stability may provide solutions for increasingly complex biomarker measurements.

Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency.

Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

With an antibody whose duty's double, a step towards ending asthma trouble?

Asthma is a debilitating chronic inflammatory disease of the airways that results in shortness of breath, wheezing and coughing, often triggered by inhalation of common allergens. The prevalence and severity of asthma has increased dramatically over the last several decades to the point where several hundred million people worldwide are affected and each year tens of thousands of individuals die prematurely from asthma symptoms. The rise in disease prevalence is particularly evident in the developed world, where childhood exposure to bacteria and viruses has decreased dramatically, while the use of antibiotics, especially early in life, and births by Caesarean section have increased significantly; correlations that support the hygiene hypothesis. Central to the pathogenesis of asthma are the roles of TH2-type cytokines, including IL (interleukin)-4, IL-5, IL-9 and IL-13, in stimulating and perpetuating an inflammatory cascade that drives detrimental changes in airway structure and function. IL-4 and IL-13, in particular, have been targets of therapeutic development because, together, they specifically promote nearly all of the clinical features of asthma. Their inflammatory functions are highly complementary, but not identical, in large part because they share cell-surface receptors and transcription factors through which they stimulate cell signalling. In this issue of the Biochemical Journal, Redpath et al. have taken advantage of the shared receptor nature of the IL-4- and IL-13-mediated inflammatory responses to generate a novel antibody to the third ectodomain (D3) of IL-13Rα1 (IL-13 receptor α1), which forms a requisite binding site for both interleukins. This antibody potently neutralizes both IL-4 and IL-13 activities in vitro. Using X-ray crystallography complemented by molecular interaction and functional analyses, they clearly define its mechanism of dual cytokine neutralization as one of molecular mimicry of the interleukin-receptor interaction. Considering the complexity of the cytokine-mediated inflammatory cascade that defines asthma pathogenesis, such therapeutics that can effectively and simultaneously neutralize the actions of multiple cytokines and may play a significantly expanded role in the treatment and prevention of asthma in the future.

Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.

The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.

Previously, we demonstrated that the γC subunit of type I IL-4 receptor was required for robust tyrosine phosphorylation of the downstream adapter protein, IRS-2, correlating with the expression of genes (ArgI, Retnla, and Chi3l3) characteristic of alternatively activated macrophages. We located an I4R-like motif (IRS-2 docking sequence) in the γC cytoplasmic domain but not in the IL-13Rα1. Thus, we predicted that the γC tail directed enhanced IRS-2 phosphorylation. To test this, IL-4 signaling responses were examined in a mutant of the key I4R motif tyrosine residue (Y325F) and different γC truncation mutants (γ285, γ308, γ318, γ323, and γFULL LENGTH (FL)) co-expressed in L-cells or CHO cells with wild-type (WT) IL-4Rα. Surprisingly, IRS-1 phosphorylation was not diminished in Y325F L-cell mutants suggesting Tyr-325 was not required for the robust insulin receptor substrate response. IRS-2, STAT6, and JAK3 phosphorylation was observed in CHO cells expressing γ323 and γFL but not in γ318 and γ285 mutants. In addition, when CHO cells expressed γ318, γ323, or γFL with IL-2Rß, IL-2 induced phospho-STAT5 only in the γ323 and γFL clones. Our data suggest that a smaller (5 amino acid) interval than previously determined is necessary for JAK3 activation/γC-mediated signaling in response to IL-4 and IL-2. Chimeric receptor chains of the γC tail fused to the IL-13Rα1 extracellular and transmembrane domain did not elicit robust IRS-2 phosphorylation in response to IL-13 suggesting that the extracellular/transmembrane domains of the IL-4/IL-13 receptor, not the cytoplasmic domains, control signaling efficiency. Understanding this pathway fully will lead to rational drug design for allergic disease.

Mapping mouse IL-13 binding regions using structure modeling, molecular docking, and high-density peptide microarray analysis.

Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation, and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13Rα1 and IL-4Rα, which is also used by IL-4. IL-13 also binds to IL-13Rα2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13Rα2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling, and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13Rα1 and IL-13Rα2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13Rα2. In addition, the identification of peptide sequences that are unique to IL-13Rα1 allowed us to generate a monoclonal antibody that selectively binds IL-13Rα1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine-receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.

The Ig-like domain of human GM-CSF receptor alpha plays a critical role in cytokine binding and receptor activation.

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.

Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay.

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Ralpha1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Ralpha1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Ralpha1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Ralpha1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.