Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential

Surface Engineering of Top7 to Facilitate Structure Determination

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination

Irreversible aggregation of alternating tetra-block-like amphiphile in water.

As a frontier topic of soft condensed matter physics, irreversible aggregation has drawn attention for a better understanding of the complex behavior of biomaterials. In this study, we have described the synthesis of an artificial amphiphilic molecule, an alternating tetra-block-like amphiphile, which was able to diversify its aggregate structure in water. The aggregated state of its aqueous dispersion was obtained by slow evaporation of the organic solvent at room temperature, and it collapsed irreversibly at ~ 50°C. By using a cryo-transmission electron microscope and a differential scanning calorimeter, it was revealed that two types of molecular nanostructures were formed and developed into submicro- and micrometer-sized fibrils in the aggregated material

Development of Small-Scale Experiments for the Education of Chemical Engineering and Its Practice for Undergraduates

A series of small-scale/micro-scale experiments used for the education of undergraduate students in chemical engineering courses have been developed. Based on the “small-scale/micro-scale” concept, the experiments were developed to provide an intuitive understanding of chemical processes, both by increasing the visibility of these chemical processes and by making the apparatus compact (desktop size). Nine experiments were developed that are relevant to the fields of thermal engineering, fluid engineering, unit operations, and reaction engineering. These experiments were introduced during the educational experiment course for undergraduates in the chemical engineering program

Analysis of CPU Loading Effect on ESD Susceptibility

Two complementary approaches are presented to help to understand how CPU loading affects the sensitivity of an electronic device to ESD (electrostatic discharge) stress. Both approaches rely on synchronized noise injection while the software is running at the desired load. One of the approaches monitors the device\u27s current consumption while the other monitors the device\u27s electromagnetic field to synchronize noise injections. These approaches revealed that as the CPU loading increases, the device becomes more active and hence more susceptible to ESD stress. Moreover, it was observed that, in each loading condition, the device randomly became susceptible. These complementary approaches enable the capturing of high/low active intervals as well as the injection of noise voltage to the desired activity, thus, allowing for the analysis of the effect of CPU loading on ESD susceptibility

Suppression of STING signaling through epigenetic silencing and missense mutation impedes DNA damage mediated cytokine production

The production of cytokines in response to DNA-damage events may be an important host defense response to help prevent the escape of pre-cancerous cells. The innate immune pathways involved in these events are known to be regulated by cellular molecules such as stimulator of interferon genes (STING), which controls type I interferon and pro-inflammatory cytokine production in response to the presence of microbial DNA or cytosolic DNA that has escaped from the nucleus. STING signaling has been shown to be defective in a variety of cancers, such as colon cancer and melanoma, actions that may enable damaged cells to escape the immunosurveillance system. Here, we report through examination of databases that STING signaling may be commonly suppressed in a greater variety of tumors due to loss-of-function mutation or epigenetic silencing of the STING/cGAS promoter regions. In comparison, RNA activated innate immune pathways controlled by RIG-I/MDA5 were significantly less affected. Examination of reported missense STING variants confirmed that many exhibited a loss-of-function phenotype and could not activate cytokine production following exposure to cytosolic DNA or DNA-damage events. Our data imply that the STING signaling pathway may be recurrently suppressed by a number of mechanisms in a considerable variety of malignant disease and be a requirement for cellular transformation

Commercial USB IC Soft-Failure Sensitivity Measurement Method and Trend Analysis

An electrostatic discharge (ESD) happening on a commercial electronic device such as at the USB interface can induce soft-failure in the IC and disturb the normal operation of the device. This paper investigates the soft-failure behaviors of 14 commercial USB devices in order to obtain an insight into the overall sensitivity trend of such systems and into the severity of different soft-failures. A new analysis method is proposed in this paper. The considered parameters in this study include: injected pulse widths, pulse rise time, current levels, USB standard of the DUTS, etc. Soft-failures started to occur at a current of around 1 A, and nearly all configurations would show errors above 6 A

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5′-<i>O</i>-<i>N</i>-(aminoacyl)­sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the <i>Escherichia coli</i> expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe–protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the <i>k</i>_cat/<i>K</i>_m value with the noncognate substrate Sal and a corresponding 48-fold decrease in the <i>k</i>_cat/<i>K</i>_m value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs