An epidermal growth factor receptor-targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance.

Effective clearance of rituximab-resistant tumor cells by breaking the mirror-symmetry of immunoglobulin G and simultaneous binding to CD55 and CD20.

Complement-dependent cytotoxicity (CDC), which eliminates aberrant target cells through the assembly and complex formation of serum complement molecules, is one of the major effector functions of anticancer therapeutic antibodies. In this study, we discovered that breaking the symmetry of natural immunoglobulin G (IgG) antibodies significantly increased the CDC activity of anti-CD20 antibodies. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in a rituximab-resistant cell line generated in-house, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance.

Engineered Human Antibody with Improved Endothelin Receptor Type A Binding Affinity, Developability, and Serum Persistence Exhibits Excellent Antitumor Potency.

Endothelin receptor A (ET), a class A G protein-coupled receptor (GPCR), is a promising tumor-associated antigen due to its close association with the progression and metastasis of many types of cancer, such as colorectal, breast, lung, ovarian, and prostate cancer. However, only small-molecule drugs have been developed as ET antagonists with anticancer effects. In a previous study, we identified an antibody (AG8) with highly selective binding to human ET through screening of a human naïve immune antibody library.

Antibody-dependent cellular cytotoxicity-null effector developed using mammalian and plant GlycoDelete platform.

Cancer therapy using immune checkpoint inhibitor antibodies has markedly shifted the paradigm of cancer treatment. However, methods completely eliminating the effector function of these signal-regulating antibodies is urgently required. The heterogeneity of glycan chains in antibodies limits their use as therapeutic agents due to their variability; thus, the development of uniform glycan chains is necessary.

An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc.

Non-Canonical Amino Acid-Based Engineering of ()-Amine Transaminase.

Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of -benzoyl phenylalanine (BpA) to engineer ()-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with BpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively.

Towards Engineering an Orthogonal Protein Translation Initiation System.

In the last two decades, methods to incorporate non-canonical amino acids (ncAAs) into specific positions of a protein have advanced significantly; these methods have become general tools for engineering proteins. However, almost all these methods depend on the translation elongation process, and strategies leveraging the initiation process have rarely been reported. The incorporation of a ncAA specifically at the translation initiation site enables the installation of reactive groups for modification at the N-termini of proteins, which are attractive positions for introducing abiological groups with minimal structural perturbations.

A human antibody against human endothelin receptor type A that exhibits antitumor potency.

Endothelin receptor A (ET), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ET nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ET antibody (AG8) exhibiting high specificity for ET in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ET using either a CHO-K1 cell line stably expressing human ET or HT-29 colorectal cancer cells, in which AG8 exhibited IC values of 56 and 51 nM, respectively.

A novel anti-c-Kit antibody-drug conjugate to treat wild-type and activating-mutant c-Kit-positive tumors.

c-Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small-cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti-c-Kit antibody-drug conjugate was developed in this study to treat wild-type and mutant c-Kit-positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (to give NN2101-DM1).

Expression of soluble recombinant human matrix metalloproteinase 9 and generation of its monoclonal antibody.

We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest.

Comparison of CD20 Binding Affinities of Rituximab Produced in Nicotiana benthamiana Leaves and Arabidopsis thaliana Callus.

Plants are promising drug-production platforms with high economic efficiency, stability, and convenience in mass production. However, studies comparing the equivalency between the original antibodies and those produced in plants are limited. Amino acid sequences that constitute the Fab region of an antibody are diverse, and the post-transcriptional modifications that occur according to these sequences in animals and plants are also highly variable.

Capture and displacement-based release of the bicarbonate anion by calix[4]pyrroles with small rigid straps.

Two-phenoxy walled calix[4]pyrroles and strapped with small rigid linkers containing pyridine and benzene, respectively, have been synthesized. H NMR spectroscopic analyses carried out in CDCl revealed that both of receptors and recognize only F and HCO among various test anions with high preference for HCO (as the tetraethylammonium, TEA salt) relative to F (as the TBA salt). The bound HCO anion was completely released out of the receptors upon the addition of F (as the tetrabutylammonium, TBA salt) as a result of significantly enhanced affinities and selectivities of the receptors for F once converted to the TEAHCO complexes.

Multi-omics characterization of the osmotic stress resistance and protease activities of the halophilic bacterium in response to salt stress.

The halophilic bacterium is well known as a promising candidate that enables the recycling of organic wastes at high salinity. However, for industrial applications of further research is required to explore the biological mechanism for maximizing the activities and productivities of this bacterium. In this study, we investigated the osmotic stress resistance and specific protease activities of in a normal-salt medium (0.

Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning.

Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed.

Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies.

The Fc region of IgG antibodies is crucial for binding to Fc receptors expressed on the surfaces of various immune leukocytes and eliciting therapeutic effector functions such as clearance of antibody-opsonized tumor cells. Despite abrogated Fc gamma receptor (FcγR) binding and therapeutic effector function in the absence of N-linked glycosylation at Asn297, the aglycosylated Fc region of IgG antibodies has bioprocessing advantages such as the absence of glycan heterogeneity and simple bacterial antibody production. Therefore, these antibodies have been comprehensively engineered as effector functional units for human therapy.

Increased Tolerance to Furfural by Introduction of Polyhydroxybutyrate Synthetic Genes to .

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes.

Low binding affinity and reduced complement-dependent cell death efficacy of ofatumumab produced using a plant system (Nicotiana benthamiana L.).

The plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the Fab region of the monoclonal antibody produced in the plant; thus, it is necessary to compare the antigen affinity of this antibody with that of the prototype. In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana, and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells.

Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs.

N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method.

Deep sequencing salivary proteins for periodontitis using proteomics.

Objectives: Saliva is a bodily fluid transuded from gingival crevice fluid and blood and contains many proteins. Proteins in saliva have been studied as markers for periodontal diseases. Mass spectrometric analysis is applied to investigate biomarker proteins that are related to periodontitis.

Peptide-Directed Photo-Cross-Linking for Site-Specific Conjugation of IgG.

Conjugation of antibody has expanded its applications in therapeutics and diagnostics, and various methods have been developed based on chemical or enzymatic reactions. However, the majority of them have focused on synthetic molecules such as small molecules, nucleic acids, or synthetic materials, but site-specific conjugation of antibody with protein cargo has rarely been demonstrated. In this Communication, we report a PEptide-DIrected Photo-cross-linking (PEDIP) reaction for site-specific conjugation of IgG with protein using an Fc-binding peptide and a photoreactive amino acid analogue, and demonstrate this method by developing an immunotoxin composed of a Her2-targeting IgG (trastuzumab) and an engineered Pseudomonas exotoxin A (PE24).

An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli.

Background: Adding new amino acids to the set of building blocks for protein synthesis expands the scope of protein engineering, and orthogonal pairs of tRNA and aminoacyl-tRNA synthetase have been developed for incorporating unnatural amino acids (UAAs) into proteins. While diverse systems have been developed to incorporate UAAs in response to the amber codon, less research has been focused on four-base codons despites their advantages. In this study, we report an efficient method to incorporate UAA in response to an AGGA codon in Escherichia coli.

Comparison of Glycopeptide Fragmentation by Collision Induced Dissociation and Ultraviolet Photodissociation.

A comparison of the fragmentation pathways of both protonated and deprotonated O-linked glycopeptides from fetuin and κ-casein obtained upon collision induced dissociation (CID) and 193 nm ultraviolet photodissociation (UVPD) in a linear ion trap is presented. A strategy using non-specific pronase digestion, zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) solid phase extraction (SPE) enrichment, and nano-liquid chromatography (nano-LC) is employed. UVPD of deprotonated glycopeptides generally produced the greatest array of fragment ions, thus affording the most diagnostic information about both glycan structure and peptide sequence.