Exploratory Prognostic Biomarkers of Complement-Mediated Thrombotic Microangiopathy (CM-TMA) in Adults with Atypical Hemolytic Uremic Syndrome (aHUS): Analysis of a Phase III Study of Ravulizumab.

BACKGROUND: Clinically validated biomarkers for monitoring of patients with complement-mediated thrombotic microangiopathy (CM-TMA) including atypical hemolytic uremic syndrome (aHUS) are unavailable. Improved characterization of biomarkers in patients with aHUS may inform treatment and monitoring for patients with CM-TMA. METHODS: This analysis used data collected from 55/56 (98.2 %) adult patients with aHUS enrolled in the global Phase III study of ravulizumab (NCT02949128). Baseline (pre-treatment) patient serum, plasma and urine biomarker levels were compared with the maximum observed levels in normal donors and evaluated for associations with pre-treatment plasma exchange/infusion and dialysis status. Biomarkers were also assessed for associations with key clinical measures at baseline and with changes at 26 and 52 weeks from treatment initiation via linear regression analyses. RESULTS: Complement-specific urine levels (factor Ba and sC5b-9) were elevated in >85 % of patients and are significantly associated with pre-treatment kidney dysfunction. Baseline levels of other evaluated biomarkers were elevated in >70 % of patients with aHUS, except for plasma sC5b-9 and serum sVCAM-1. Lower levels of urine complement markers at baseline are significantly associated with improvements in total urine protein and estimated glomerular filtration rate at 26 and 52 weeks of treatment. Clinical assessment of complement activation by a receiver operating characteristic analysis of Ba and sC5b-9 was more sensitive and specific in urine matrix than plasma. CONCLUSION: This analysis identified a set of biomarkers that may show utility in the prognosis of CM-TMA, including their potential for measuring and predicting response to anti-C5 therapy. Further studies are required to enhance patient risk stratification and improve management of these vulnerable patients. CLINICAL TRIALS REGISTRATION: NCT02949128, ClinicalTrials.gov.

Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering.

Genetic selection from libraries expressing proteins with randomized amino acid segments is a powerful approach to identify proteins with novel biological activities. Here, we assessed the utility of deep DNA sequencing to characterize the composition, diversity, size and stability of such randomized libraries. We used 454 pyrosequencing to sequence a retroviral library expressing small proteins with randomized transmembrane domains. Despite the potential for unintended random mutagenesis during its construction, the overall hydrophobic composition and diversity of the proteins encoded by the sequenced library conformed well to its design. In addition, our sequencing results allowed us to calculate a more accurate estimate of the number of different proteins encoded by the library and suggested that the traditional methods for estimating the size of randomized libraries may overestimate their true size. Our results further demonstrated that no significant genetic bottlenecks exist in the methods used to express complex retrovirus libraries in mammalian cells and recover library sequences from these cells. These findings suggest that deep sequencing can be used to determine the quality and content of other libraries with randomized segments and to follow individual sequences during selection.

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.

The ankyrin repeat as molecular architecture for protein recognition.

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.

Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations.

Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein.