A novel conditional active biologic anti-EpCAM x anti-CD3 bispecific antibody with synergistic tumor selectivity for cancer immunotherapy.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays several roles in cancer biology. EpCAM is an attractive therapeutic target because of its expression in most solid tumors. However, targeting EpCAM has been challenging because it is also highly expressed in normal epithelial tissues. Initial attempts to develop EpCAM-specific T-cell engagers were unsuccessful due to severe cytokine release effects, as well as serious on-target, off-tumor drug-related toxicities. We developed novel, conditionally active biological (CAB) bispecific antibodies that bind to both EpCAM and CD3 in an acidic tumor microenvironment. In healthy tissues, binding to EpCAM and CD3 is greatly reduced by a novel, dual CAB selection, where each binding domain is independently blocked by the presence of physiological chemicals known as Protein-associated Chemical Switches (PaCS). The CAB anti-EpCAM T-cell engagers displayed the anticipated bispecific binding properties and mediated the potent lysis of EpCAM-positive cancer cell lines through the recruitment of T cells in the tumor microenvironment. Xenograft studies showed that the efficacy of CAB bispecific antibodies is similar to that of a non-CAB anti-EpCAM bispecific antibody, but they have markedly reduced toxicity in non-human primates, indicating an unprecedentedly widened therapeutic index of over 100-fold. These preclinical results indicate that the dual CAB bispecific antibody is potentially both a powerful and safe therapeutic platform and a promising T cell-engaging treatment for patients with EpCAM-expressing tumors.

Development of a novel conditionally active EpCAM-specific T-cell engager with enhanced safety and tolerability for treatment of solid tumors.

Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches.

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

A ketogenic diet combined with exercise alters mitochondrial function in human skeletal muscle while improving metabolic health.

Animal data indicate that ketogenic diets are associated with improved mitochondrial function, but human data are lacking. We aimed to characterize skeletal muscle mitochondrial changes in response to a ketogenic diet combined with exercise training in healthy individuals. Twenty-nine physically active adults completed a 12-wk supervised exercise program after self-selection into a ketogenic diet (KD, n = 15) group or maintenance of their habitual mixed diet (MD, n = 14). Measures of metabolic health and muscle biopsies (vastus lateralis) were obtained before and after the intervention. Mitochondria were isolated from muscle and studied after exposure to carbohydrate (pyruvate), fat (palmitoyl-l-carnitine), and ketone (ß-hydroxybutyrate+acetoacetate) substrates. Compared with MD, the KD resulted in increased whole body resting fat oxidation (P < 0.001) and decreased fasting insulin (P = 0.019), insulin resistance [homeostatic model assessment of insulin resistance (HOMA-IR), P = 0.022], and visceral fat (P < 0.001). The KD altered mitochondrial function as evidenced by increases in mitochondrial respiratory control ratio (19%, P = 0.009), ATP production (36%, P = 0.028), and ATP/H2O2 (36%, P = 0.033) with the fat-based substrate. ATP production with the ketone-based substrate was four to eight times lower than with other substrates, indicating minimal oxidation. The KD resulted in a small decrease in muscle glycogen (14%, P = 0.035) and an increase in muscle triglyceride (81%, P = 0.006). These results expand our understanding of human adaptation to a ketogenic diet combined with exercise. In conjunction with weight loss, we observed altered skeletal muscle mitochondrial function and efficiency, an effect that may contribute to the therapeutic use of ketogenic diets in various clinical conditions, especially those associated with insulin resistance.

A Pre-Workout Supplement of Ketone Salts, Caffeine, and Amino Acids Improves High-Intensity Exercise Performance in Keto-Naïve and Keto-Adapted Individuals.

Background: Acute ingestion of ketone supplements alters metabolism and potentially exercise performance. No studies to date have evaluated the impact of co-ingestion of ketone salts with caffeine and amino acids on high intensity exercise performance, and no data exists in Keto-Adapted individuals.Methods: We tested the performance and metabolic effects of a pre-workout supplement containing beta-hydroxybutyrate (BHB) salts, caffeine, and amino acids (KCA) in recreationally-active adults habitually consuming a mixed diet (Keto-Naïve; n = 12) or a ketogenic diet (Keto-Adapted; n = 12). In a randomized and balanced manner, subjects consumed either the KCA consisting of ∼7 g BHB (72% R-BHB and 28% S-BHB) with ∼100 mg of caffeine, and amino acids (leucine and taurine) or Water (control condition) 15 minutes prior to performing a staged cycle ergometer time to exhaustion test followed immediately by a 30 second Wingate test.Results: Circulating total BHB concentrations increased rapidly after KCA ingestion in KN (154 to 732 µM) and KA (848 to 1,973 µM) subjects and stayed elevated throughout recovery in both groups. Plasma S-BHB increased >20-fold 15 minutes after KCA ingestion in both groups and remained elevated throughout recovery. Compared to Water, KCA ingestion increased time to exhaustion 8.3% in Keto-Naïve and 9.8% in Keto-Adapted subjects (P < 0.001). There was no difference in power output during the Wingate test between trials. Peak lactate immediately after exercise was higher after KCA (∼14.9 vs 12.7 mM).Conclusion: These results indicate that pre-exercise ingestion of a moderate dose of R- and S-BHB salts combined with caffeine, leucine and taurine improves high-intensity exercise performance to a similar extent in both Keto-Adapted and Keto-Naïve individuals.

Extended Ketogenic Diet and Physical Training Intervention in Military Personnel.

INTRODUCTION: Ketogenic diets (KDs) that elevate ketones into a range referred to as nutritional ketosis represent a possible nutrition approach to address the emerging physical readiness and obesity challenge in the military. An emerging body of evidence demonstrates broad-spectrum health benefits attributed to being in nutritional ketosis, but no studies have specifically explored the use of a KD in a military population using daily ketone monitoring to personalize the diet prescription. MATERIALS AND METHODS: To evaluate the feasibility, metabolic, and performance responses of an extended duration KD, healthy adults (n = 29) from various military branches participated in a supervised 12-wk exercise training program. Fifteen participants self-selected to an ad libitum KD guided by daily measures of capillary blood ketones and 14 continued their normal mixed diet (MD). A battery of tests were performed before and after the intervention to assess changes in body mass, body composition, visceral fat, liver fat, insulin sensitivity, resting energy metabolism, and physical performance. RESULTS: All KD subjects were in nutritional ketosis during the intervention as assessed by daily capillary beta-hydroxybutyrate (ßHB) (mean ßHB 1.2 mM reported 97% of all days) and showed higher rates of fat oxidation indicative of keto-adaptation. Despite no instruction regarding caloric intake, the KD group lost 7.7 kg body mass (range -3.5 to -13.6 kg), 5.1% whole-body percent fat (range -0.5 to -9.6%), 43.7% visceral fat (range 3.0 to -66.3%) (all p < 0.001), and had a 48% improvement in insulin sensitivity; there were no changes in the MD group. Adaptations in aerobic capacity, maximal strength, power, and military-specific obstacle course were similar between groups (p > 0.05). CONCLUSIONS: US military personnel demonstrated high adherence to a KD and showed remarkable weight loss and improvements in body composition, including loss of visceral fat, without compromising physical performance adaptations to exercise training. Implementation of a KD represents a credible strategy to enhance overall health and readiness of military service members who could benefit from weight loss and improved body composition.

Quantification of Human Central Adipose Tissue Depots: An Anatomically Matched Comparison Between DXA and MRI.

Excess visceral adipose tissue (VAT) and VAT volume relative to subcutaneous adipose tissue (SAT) are associated with elevated health risks. This study compares fat measurements by dual-energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI). In total, 21 control subjects (Control) and 16 individuals with metabolic syndrome (MetSyn) were scanned by DXA and MRI. The region measured by MRI was matched to the android region defined by DXA, and MRI reproducibility was also evaluated. In addition, liver fat fraction was quantified via MRI and whole-body fat by DXA. VAT measurements are interchangeable between DXA and MRI in the Control (R = 0.946), MetSyn (R = 0.968), and combined cohort (R = 0.983). VAT/SAT ratio did not differ in the Control group (P = .10), but VAT/SAT ratio measured by DXA was significantly higher in the MetSyn group (P < .01) and the combined (P = .03) cohort. Intraobserver (ICC = 0.998) and interobserver (ICC = 0.977) reproducibility of MRI VAT measurements was excellent. Liver fat fraction by MRI was higher (P = .001) in MetSyn (12.4% ± 7.6%) than in controls (2.6% ± 2.2%), as was whole-body fat percentage by DXA (P = .001) between the MetSyn (42.0% ± 8.1%) and Control groups (26.7% ± 6.9%). DXA and MRI VAT are interchangeable when measured over an anatomically matched region of the abdomen, while SAT and VAT/SAT ratio differ between the 2 modalities.

Neutralization of Clostridium difficile toxin A using antibody combinations.

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.

A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut.

A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different beta-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes beta-1,4-linked mannan substrates, while the second catalytic site hydrolyzes beta-1,4-linked xylan and beta-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression.

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.

High-throughput cultivation of microorganisms using microcapsules.

This chapter describes a universal and novel method that provides access to the immense reservoir of untapped microbial diversity by cultivation. This technique uses microcapsules to encapsulate single cells combined with parallel microbial cultivation under low nutrient flux conditions. Under these conditions, single encapsulated cells grow and form microcolonies within the microcapsules. Flow cytometry is used as a sensitive tool to detect growth within the microcapsules. Microcapsules that contain microcolonies (originated from a single encapsulated cell) are sorted individually into microtiter dishes containing organic-rich medium. This high-throughput cultivation can provide more than 10,000 bacterial and fungal isolates per environmental sample.

Genome streamlining in a cosmopolitan oceanic bacterium.

The SAR11 clade consists of very small, heterotrophic marine alpha-proteobacteria that are found throughout the oceans, where they account for about 25% of all microbial cells. Pelagibacter ubique, the first cultured member of this clade, has the smallest genome and encodes the smallest number of predicted open reading frames known for a free-living microorganism. In contrast to parasitic bacteria and archaea with small genomes, P. ubique has complete biosynthetic pathways for all 20 amino acids and all but a few cofactors. P. ubique has no pseudogenes, introns, transposons, extrachromosomal elements, or inteins; few paralogs; and the shortest intergenic spacers yet observed for any cell.

Global proteome discovery using an online three-dimensional LC-MS/MS.

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.

Comparative metagenomics of microbial communities.

The species complexity of microbial communities and challenges in culturing representative isolates make it difficult to obtain assembled genomes. Here we characterize and compare the metabolic capabilities of terrestrial and marine microbial communities using largely unassembled sequence data obtained by shotgun sequencing DNA isolated from the various environments. Quantitative gene content analysis reveals habitat-specific fingerprints that reflect known characteristics of the sampled environments. The identification of environment-specific genes through a gene-centric comparative analysis presents new opportunities for interpreting and diagnosing environments.

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement.

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62 degrees C for 1 h and 27% of its initial activity after 10 min at 85 degrees C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

Exploring nitrilase sequence space for enantioselective catalysis.

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.