Directed evolution methods for overcoming trade‐offs between protein activity and stability

Engineered proteins are being widely developed and employed in applications ranging from enzyme catalysts to therapeutic antibodies. Directed evolution, an iterative experimental process composed of mutagenesis and library screening, is a powerful technique for enhancing existing protein activities and generating entirely new ones not observed in nature. However, the process of accumulating mutations for enhanced protein activity requires chemical and structural changes that are often destabilizing, and low protein stability is a significant barrier to achieving large enhancements in activity during multiple rounds of directed evolution. Here we highlight advances in understanding the origins of protein activity/stability trade‐offs for two important classes of proteins (enzymes and antibodies) as well as innovative experimental and computational methods for overcoming such trade‐offs. These advances hold great potential for improving the generation of highly active and stable proteins that are needed to address key challenges related to human health, energy and the environment.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154495/1/aic16814_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154495/2/aic16814.pd

Occupational noise exposure and risk of hypertension in an industrial workforce

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140051/1/ajim22775_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/140051/2/ajim22775.pd

Nature inspired antibody design and optimization

The biotech industry has seen an explosion in the development of therapeutic antibodies in the last decade. The advantages of antibodies as therapeutics – namely their high affinity, specificity, potency, stability, manufacturability and low toxicity – are compelling. Nevertheless, there are many challenges associated with antibody discovery and development that require key technical advances to improve the rational and reliable generation of potent antibody therapeutics. We have made three key discoveries that address some of these fundamental challenges related to the design and selection of antibodies with high affinity, specificity, stability and solubility. First, we find that the accumulation of affinity-enhancing mutations in the complementaritydetermining regions (CDRs) during affinity maturation is often a destabilizing process. Surprisingly, mutations that enhance antibody binding affinity are commonly destabilizing. Second, we have developed novel yeast surface display methods for co-evolving antibody affinity and stability to address the general problem of antibody destabilization during affinity maturation. Our approach simultaneously evaluates antibody binding to both antigen and a conformational ligand that acts as a folding sensor to rapidly identify sets of mutations that promote both high antibody affinity and stability. This methodology has enabled us to identify novel compensatory mutations that offset the destabilizing effects of affinity-enhancing mutations and lead to affinitymaturated antibodies with high thermodynamic stability. Interestingly, our directed evolution method appears to mimic some aspects of natural antibody evolution, as natural antibodies also accumulate similar types of compensatory mutations to maintain thermodynamic stability during in vivo affinity maturation. Third, we have developed novel antibody library design and selection methods for generating antibodies with high specificity. It is common for antibody specificity to be compromised during in vitro affinity maturation. We have developed innovative methods for designing antibody libraries based on natural antibody diversity to simultaneously sample residues at many sites in the CDRs and framework regions that are most likely to promote high specificity. By coupling these nature-inspired antibody libraries with novel positive and negative selection methods, we have isolated antibodies with specificities that rival those of natural antibodies and which are much higher than typical antibodies identified using in vitro selection methods. Interestingly, we find that antibodies with improved specificity also possess excellent biophysical properties, including high solubility and stability. We are currently using computational methods to understand how rare antibody variants are able to maintain high specificity and stability during affinity maturation. Our long-term goal is to develop systematic and robust design methods to rapidly generate and optimize antibodies for use in a range of diagnostic and therapeutic applications

Publisher Correction: An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Sensitive detection of glucagon aggregation using amyloid fibril‐specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (20 times more sensitive than detection using a conventional, amyloid‐specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (20 times more sensitive than conventional methods for detecting glucagon fibrils.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150615/1/bit26994_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150615/2/bit26994.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150615/3/bit26994-sup-0001-Supporting_Information__submission_.pd

Development of a Chitosan-Based Biofoam: Application to the Processing of a Porous Ceramic Material

Developing biofoams constitutes a challenging issue for several applications. The present study focuses on the development of a chitosan-based biofoam. Solutions of chitosan in acetic acid were dried under vacuum to generate foams with high-order structures. Chitosan concentration influenced significantly the morphology of developed porosity and the organization of pores in the material. Physico-chemical characterizations were performed to investigate the effects of chitosan concentration on density and thermal conductivity of foams. Even if chitosan-based biofoams exhibit interesting insulating properties (typically around 0.06 W·m−1·K−1), it has been shown that their durabilities are limited when submitted to a wet media. So, a way of application consists to elaborate a ceramic material with open porosity from a slurry prepared with an organic solvent infiltrating the porous network of the foam

Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis

Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8- and interferon-γ (IFN-γ)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-γ and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-γ) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and inactive (n = 2) JRA. A subpopulation of polyarticular JRA neutrophils are in a chronic, activated state, a state that persists when the disease is well controlled pharmacologically. Furthermore, polyarticular JRA neutrophils exhibit an intrinsic defect in the regulation of metabolic oscillations and superoxide ion production. Our data are consistent with the hypothesis that neutrophils play an essential role in the pathogenesis of polyarticular JRA

An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.

The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered Fc domains that confer a longer circulation half-life by virtue of more favorable pH-dependent binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically to recapitulate all the key processes relevant to human antibody persistence in circulation, namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which are important for the clearance of target pathogenic cells and also has favorable developability

Fine-mapping identifies multiple prostate cancer risk loci at 5p15, one of which associates with TERT expression

Associations between single nucleotide polymorphisms (SNPs) at 5p15 and multiple cancer types have been reported. We have previously shown evidence for a strong association between prostate cancer (PrCa) risk and rs2242652 at 5p15, intronic in the telomerase reverse transcriptase (TERT) gene that encodes TERT. To comprehensively evaluate the association between genetic variation across this region and PrCa, we performed a fine-mapping analysis by genotyping 134 SNPs using a custom Illumina iSelect array or Sequenom MassArray iPlex, followed by imputation of 1094 SNPs in 22 301 PrCa cases and 22 320 controls in The PRACTICAL consortium. Multiple stepwise logistic regression analysis identified four signals in the promoter or intronic regions of TERT that independently associated with PrCa risk. Gene expression analysis of normal prostate tissue showed evidence that SNPs within one of these regions also associated with TERT expression, providing a potential mechanism for predisposition to disease

Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies

Researchers rely largely on antibodies to measure the abundance, activity, and localization of a protein, information that provides critical insight into both normal and pathological cellular functions. However, antibodies are not always reliable or universally valid for the methods in which they are used; in particular, the reliability of commercial antibodies against RAS is highly variable. Waters et al . rigorously assessed 22 commercially available RAS antibodies for their utility to detect the distinct RAS isoforms in various cell types and for their use in specific analytical methods. Their findings show how reliably one can interpret the data acquired from each reagent