Optimization of E. coli SoluProTM using synthetic biology to a generate a high performance chassis microbe for scalable production of protein therapeutics

E. coli is a historically important research tool for early phase discovery and development of protein therapeutics. Nevertheless, Chinese Hamster Ovary (CHO) and other mammalian cell lines are the predominant production hosts for current generation antibody and antibody fragment production. Microbial hosts such as E. coli are used to produce a minority of approved biologic drugs relative to mammalian cell lines, due in part to perceived limitations in protein solubility and quality related to the complexities of protein folding, maturation, host specific post-translational modifications, as well as regulatory considerations. However, recent advances in our understanding of microbial biology and synthetic biology have enabled rapid progress to be made in the development of microbial cell lines that exceed the performance of best-in-class mammalian cell lines. AbSci has developed a functional reconstruction of the protein production environment of the eukaryotic endoplasmic reticulum in E. coli, which includes a semi-oxidized cytoplasm that facilitates appropriate protein folding and disulfide bond formation. Within the physiological context of E. coli SoluPro™, a best-in-class synthetic biology strategy that modulates rates of gene expression, protein expression, and protein folding using a plasmid-based design architecture have been validated as a strategy to produce soluble, high titer and quality protein biologics. Following construction of millions of plasmid variants using a pooled DNA construction library approach, plasmids are screened in vivo for improvements in protein titer and quality using a fluorescence activated cell sorting (FACS)-mediated antigen binding assay. Next generating sequencing (NGS) is used to identify genotypes enriched within populations of cells with enhanced antigen-binding properties. Secondary assays are performed to validate strain improvements identified by flow cytometry, including an orthogonal screening of antibody-mediated antigen-binding in cell lysates, as well as advanced Mass Spectrometry methods to quantify disulfide bond formation and other protein quality attributes. This strategy has enabled rapid identification of plasmid designs for soluble production of full-length antibodies and antibody fragments that can be scaled to multigram quantities of product in bioreactor fermentations of 48 hours or less. Additional optimization of the E. coli SoluPro™ chassis is being tailored to further improve folding and maturation of additional classes of complex therapeutic proteins. The ease of use of E. coli and technical robustness of our high-throughput discovery and optimization workflow enables AbSci to rapidly identify key conditions for heterologous protein production and identify protein folding solutions conditions that can exceed Gram level quantities of soluble protein with less than three months of strain optimization effort

Biopsy-Controlled Non-Invasive Quantification of Collagen Type VI in Kidney Transplant Recipients:A Post-Hoc Analysis of the MECANO Trial

The PRO-C6 assay, a reflection of collagen type VI synthesis, has been proposed as a non-invasive early biomarker of kidney fibrosis. We aimed to investigate cross-sectional and longitudinal associations between plasma and urine PRO-C6 and proven histological changes after kidney transplantation. The current study is a post-hoc analysis of 94 participants of the MECANO trial, a 24-month prospective, multicenter, open-label, randomized, controlled trial aimed at comparing everolimus-based vs. cyclosporine-based immunosuppression. PRO-C6 was measured in plasma and urine samples collected 6 and 24 months post-transplantation. Fibrosis was evaluated in biopsies collected at the same time points by Banff interstitial fibrosis/tubular atrophy (IF/TA) scoring and collagen staining (Picro Sirius Red; PSR); inflammation was evaluated by the tubulo-interstitial inflammation score (ti-score). Linear regression analyses were performed. Six-month plasma PRO-C6 was cross-sectionally associated with IF/TA score (Std. beta = 0.34), and prospectively with 24-month IF/TA score and ti-score (Std. beta = 0.24 and 0.23, respectively) (p <0.05 for all). No significant associations were found between urine PRO-C6 and any of the biopsy findings. Fibrotic changes and urine PRO-C6 behaved differentially over time according to immunosuppressive therapy. These results are a first step towards non-invasive fibrosis detection after kidney transplantation by means of collagen VI synthesis measurement, and further research is required

Mutacin 1140 Lantibiotic Variants Are Efficacious Against Clostridium difficile Infection

Lantibiotics offer an untapped pipeline for the development of novel antibiotics to treat serious Gram-positive (+) infections including Clostridium difficile. Mutacin 1140 (MU1140) is a lantibiotic produced by Streptococcus mutans and acts via a novel mechanism of action, which may limit the development of resistance. This study sought to identify a lead compound for the treatment of C. difficile associated diarrhea (CDAD). Compounds were selected from a saturation mutagenesis library of 418 single amino acid variants of MU1140. Compounds were produced by small scale fermentation, purified, characterized and then subjected to a panel of assays aimed at identifying the best performers. The screening assays included: in vitro susceptibility testing [MIC against Micrococcus luteus, Clostridium difficile, vancomycin-resistant enterococci (VRE), Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium phlei, and Pseudomonas aeruginosa; cytotoxicity screening on HepG2 hepatocytes; in vitro pharmacological profiling with the Safety Screen 44TM, metabolic and chemical stability in biologically relevant fluids (FaSSGF, FaSSIF and serum); and efficacy in vivo]. Several lantibiotic compounds had better MIC against C. difficile, compared to vancomycin, but not against other bacterial species tested. The Safety Screen 44TMin vitro pharmacological profiling assay suggested that this class of compounds has relatively low overall toxicity and that compound OG253 (MU1140, Phe1Ile) is not likely to present inadvertent off-target effects, as evidenced by a low promiscuity score. The in vitro cytotoxicity assay also indicated that this class of compounds was characterized by low toxicity; the EC50 of OG253 was 636 mg/mL on HepG2 cells. The half-life in simulated gastric fluid was &gt;240 min. for all compound tested. The stability in simulated intestinal fluid ranged between a half-life of 5 min to &gt;240 min, and paralleled the half-life in serum. OG253 ultimately emerged as the lead compound based on superior in vivo efficacy along with an apparent lack of relapse in a hamster model of infection. The lessons learned from this report are applicable to therapeutic lanthipeptides in general and may assist in the design of novel molecules with improved pharmacological, therapeutic and physicochemical profiles. The data presented also support the continued clinical development of OG253 as a novel antibiotic against CDAD that could prevent recurrence of the infection

OG716: Designing a fit-for-purpose lantibiotic for the treatment of <i>Clostridium difficile</i> infections

<div><p>Lantibiotics continue to offer an untapped pipeline for the development of novel antibiotics. We report here the discovery of a novel lantibiotic for the treatment of <i>C</i>. <i>difficile</i> infection (CDI). The leads were selected from a library of over 300 multiple substitution variants of the lantibiotic Mutacin 1140 (MU1140). Top performers were selected based on testing for superior potency, solubility, manufacturability, and physicochemical and/or metabolic stability in biologically-relevant systems. The best performers <i>in vitro</i> were further evaluated orally in the Golden Syrian hamster model of CDAD. <i>In vivo</i> testing ultimately identified OG716 as the lead compound, which conferred 100% survival and no relapse at 3 weeks post infection. MU1140-derived variants are particularly attractive for further clinical development considering their novel mechanism of action.</p></div

Structural features of MU1140 and select lead compounds.

<p>Panel (A) depicts the primary amino acid sequence of MU1140. The second generation of MU1140 variants designed in the current study focused on the residues highlighted in gray. Panel (B) depicts the structure of unusual amino acids. Panel (C) tabulates the substitutions of lead compounds carried through <i>in vivo</i> efficacy studies. Legend: amino acids, AA.</p

MIC characteristics of selected compounds against <i>C</i>. <i>difficile</i>¹.

<p>MIC characteristics of selected compounds against <i>C</i>. <i>difficile</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#t003fn001" target="_blank">¹</a>.</p

Hamster CDAD study design.

<p>Hamster CDAD study design.</p

Cytochrome P450–catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N′-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase–generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O2

Efficacy of lead compounds assessed <i>in vivo</i>.

<p>Golden Syrian hamsters (N = 6 per group) were infected on Day 1 and received a single subcutaneous injection of Clindamycin (10 mg/Kg) on Day 2. Test compounds at 20 mg/Kg in 5% mannitol were administered by oral gavage 3 times per day (TID), starting on Day 2 at 18 hours after Clindamycin treatment, for 5 consecutive days (Days 2 through 6). Vancomycin (positive control) was administered at 20 mg/kg QD in parallel, and the infection control group was dosed with vehicle alone. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#sec002" target="_blank">Materials and Methods</a></b>for details.</p