Rapid and Convenient Single-Chain Variable Fragment-Employed Electrochemical C-Reactive Protein Detection System.

Although IgG-free immunosensors are in high demand owing to ethical concerns, the development of convenient immunosensors that alternatively integrate recombinantly produced antibody fragments, such as single-chain variable fragments (scFvs), remains challenging. The low affinity of antibody fragments, unlike IgG, caused by monovalent binding to targets often leads to decreased sensitivity. We improved the affinity owing to the bivalent effect by fabricating a bivalent antibody-enzyme complex (AEC) composed of two scFvs and a single glucose dehydrogenase, and developed a rapid and convenient scFv-employed electrochemical detection system for the C-reactive protein (CRP), which is a homopentameric protein biomarker of systemic inflammation. The development of a point-of-care testing (POCT) system is highly desirable; however, no scFv-based CRP-POCT immunosensors have been developed. As expected, the bivalent AEC showed higher affinity than the single scFv and contributed to the high sensitivity of CRP detection. The electrochemical CRP detection using scFv-immobilized magnetic beads and the bivalent AEC as capture and detection antibodies, respectively, was achieved in 20 min without washing steps in human serum and the linear range was 1-10 nM with the limit of detection of 2.9 nM, which has potential to meet the criteria required for POCT application in rapidity, convenience, and hand-held detection devices without employing IgGs.

The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases.

We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use ß-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 Å from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1-4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.

Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA-Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element.

DNA-protein complexes are attractive components with broad applications in various research fields, such as DNA aptamer-enzyme complexes as biosensing elements. However, noncovalent DNA-protein complexes often decrease detection sensitivity because they are highly susceptible to environmental conditions. In this study, we developed a versatile DNA-protein covalent-linking patch (D-Pclip) for fabricating covalent and stoichiometric DNA-protein complexes. We comprehensively explored the database to determine the DNA-binding ability of the candidates and selected UdgX as the only uracil-DNA glycosylase known to form covalent bonds with DNA via uracil, with a binding efficiency >90%. We integrated a SpyTag/SpyCatcher protein-coupling system into UdgX to create a universal and convenient D-Pclip. The usability of D-Pclip was shown by preparing a stoichiometric model complex of a hemoglobin (Hb)-binding aptamer and glucose oxidase (GOx) by mixing at 4 °C. The prepared aptamer-GOx complexes detected Hb in a dose-dependent manner within the clinically required detection range in buffer and human serum without any washing procedures. D-Pclip covalently connects any uracil-inserted DNA sequence and any SpyCatcher-fused protein stoichiometrically; therefore, it has a high potential for various applications.

Cancer therapeutic trispecific antibodies recruiting both T and natural killer cells to cancer cells.

T cells and natural killer (NK) cells are major effector cells recruited by cancer therapeutic bispecific antibodies; however, differences in the populations of these cells in individual tumors limit the general use of these antibodies. In the present study, trispecific antibodies were created, namely T cell and NK cell engagers (TaKEs), that recruit both T cells and NK cells. Notably, three Fc­fused TaKEs were designed, TaKE1­Fc, TaKE2­Fc and TaKE3­Fc, using variable fragments targeting the epidermal growth factor receptor on tumor cells, CD3 on T cells, and CD16 on NK cells. Among them, TaKE1­Fc was predicted to form a circular tetrabody­like configuration and exhibited the highest production and greatest cancer growth inhibitory effects. TaKE1 was prepared from TaKE1­Fc by digesting the Fc region for further functional evaluation. The resulting TaKE1 exhibited trispecificity via its ability to bind cancer cells, T cells and NK cells, as well as comparable or greater cancer growth inhibitory effects to those of two bispecific antibodies that recruit T cells and NK cells, respectively. A functional trispecific antibody with the potential to exert strong therapeutic effects independent of T cell and NK cell populations was developed.

Functional integration of protein A binding ability to antibody fragments for convenient and tag-free purification.

Although the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.

Construction of IgG-Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction.

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG-Fab2-type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG-Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG-Fab2 type bsAb will expand the bsAb production method.

Incorporation of a repeated polypeptide sequence in therapeutic antibodies as a universal masking procedure: A case study of T cell-engaging bispecific antibodies.

Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies.

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System.

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method.

Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer.

Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused Oplophorus luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals (R2 = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.

Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

Strategies to simplify operation procedures for applying labeled antibody-based immunosensors to point-of-care testing.

Point-of-care testing (POCT) is an ideal testing format for the rapid and on-site detection of analytes in patients, and facilitates disease diagnosis and monitoring. Molecular recognition elements are required for the specific detection of analytes, and biosensors that use antibodies as the molecular recognition elements are called immunosensors. Traditional immunosensors such as sandwich enzyme-linked immunosorbent assay (ELISA) require complicated procedures to form immunocomplexes consisting of detection antibodies, analytes, and capture antibodies. They also require long incubation times, washing procedures, and large and expensive specialized equipment that must be operated by laboratory technicians. Immunosensors for POCT should be systems that use relatively small pieces of equipment and do not require special training. In this review, to help in the construction of immunosensors for POCT, we have summarized the recently reported strategies for simplifying the operation, incubation, and washing procedures. We focused on the optical and electrochemical detection principles of immunosensors, compared the strategies for operation, sensitivity, and detection devices and discussed the ideal system. Combining detection devices that can be fabricated inexpensively and strategies that enable simplification of operation procedures and enhance sensitivities will contribute to the development of immunosensors for POCT.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Fabrication of Fragment Antibody-Enzyme Complex as a Sensing Element for Immunosensing.

Antibody-enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific targets and catalytic activity to gain signals, particularly oxidoreductases, which can be integrated into rapid and sensitive electrochemical measurements. The development of AECs using fragment antibodies rather than intact antibodies, such as immunoglobulin G (IgG), has attracted attention for overcoming the ethical and cost issues associated with the production of intact antibodies. Conventionally, chemical conjugation has been used to fabricate AECs; however, controlling stoichiometric conjugation using this method is difficult. To prepare homogeneous AECs, methods based on direct fusion and enzymatic conjugation have been developed, and more convenient methods using Catcher/Tag systems as coupling modules have been reported. In this review, we summarize the methods for fabricating AECs using fragment antibodies developed for sensing applications and discuss the advantages and disadvantages of each method.

Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria.

BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

Transient potentiometry based d-serine sensor using engineered d-amino acid oxidase showing quasi-direct electron transfer property.

d-Serine biosensing has been extensively reported based on enzyme sensors using flavin adenine dinucleotide (FAD) -dependent d-amino acid oxidase (DAAOx), based on the monitoring of hydrogen peroxide generated by the enzymatic reaction, which is affected by dissolved oxygen concentration in the measurement environment in in vivo use. Here we report a novel sensing principle for d-serine, transient potentiometry based d-serine sensor using engineered DAAOx showing quasi-direct electron transfer (DET) property. DAAOx Gly52Val mutant, revealed to possess dye-mediated dehydrogenase activity using artificial synthetic electron acceptors, while its oxidase activity was negligible. The enzyme was immobilized on electrode and was modified with amine-reactive phenazine ethosulfate, resulted an enzyme electrode showing quasi-DET type response. Although OCP based monitoring took more than several minutes to obtain steady state OCP value, the time dependent OCP change monitoring, transient potentiometry, provided rapid and sensitive sensor signals. While dOCP/dt based monitoring was suitable for sensing with longer than 5 s time resolution with d-serine concentration range between 0.5 mM and 5 mM, dOCP/d t based monitoring is suitable for d-serine monitoring with much shorter time resolution (less than 1 s) with high sensitivity with wider dynamic range (20 µM-30 mM). The maximum dOCP/d t was -39.2 ± 2.0 mV/s1/2, the Km(app) was 1.9 mM, and the lower limit of detection was 20 µM. In addition, d-serine monitoring was also possible in the artificial cerebrospinal fluid. The transient potentiometry based sensing reported in this study will be further utilized to realize miniaturized, continuous, real-time, in vivo sensor for d-serine monitoring.

Development of a POCT type insulin sensor employing anti-insulin single chain variable fragment based on faradaic electrochemical impedance spectroscopy under single frequency measurement.

To improve glycemic control managed through insulin administration, recent studies have focused on developing hand-held point-of-care testing (POCT) electrochemical biosensors for insulin measurement. Amongst them, anti-insulin IgG-based sensors show promise in detecting insulin with high specificity and sensitivity. However, fabrication of electrochemical sensors with IgG antibodies can prove challenging because of their larger molecular size. To overcome these limitations, this study focuses on utilizing the anti-insulin single chain variable fragment (scFv) as a biosensing molecule with single-frequency faradaic electrochemical impedance spectroscopy (EIS). By comparing two different immobilization methods, covalent conjugation via succinimidyl ester and non-covalent poly-histidine chelation, we demonstrated effective modification of the electrode surface with anti-insulin scFv, while retaining its specific recognition toward insulin. Sensor performance was confirmed via the concentration-dependent faradaic electrochemical impedance change using potassium ferricyanide as a redox probe. The optimal frequency for measurement was determined to be the peak slope of the calculated impedance correlation with respect to frequency. Based on the identified optimized frequency, we performed single-frequency measurement of insulin within a concentration range of 10 pM-100 nM. This study can aid in developing a future point-of-care sensor which rapidly and sensitively measures insulin across a dynamic range of physiological concentrations, with label-free detection.

T Cell Bispecific Antibodies: An Antibody-Based Delivery System for Inducing Antitumor Immunity.

As a breakthrough immunotherapy, T cell bispecific antibodies (T-BsAbs) are a promising antibody therapy for various kinds of cancer. In general, T-BsAbs have dual-binding specificity to a tumor-associated antigen and a CD3 subunit forming a complex with the TCR. This enables T-BsAbs to crosslink tumor cells and T cells, inducing T cell activation and subsequent tumor cell death. Unlike immune checkpoint inhibitors, which release the brake of the immune system, T-BsAbs serve as an accelerator of T cells by stimulating their immune response via CD3 engagement. Therefore, they can actively redirect host immunity toward tumors, including T cell recruitment from the periphery to the tumor site and immunological synapse formation between tumor cells and T cells. Although the low immunogenicity of solid tumors increases the challenge of cancer immunotherapy, T-BsAbs capable of immune redirection can greatly benefit patients with such tumors. To investigate the detailed relationship between T-BsAbs delivery and their T cell redirection activity, it is necessary to determine how T-BsAbs deliver antitumor immunity to the tumor site and bring about tumor cell death. This review article discusses T-BsAb properties, specifically their pharmacokinetics, redirection of anticancer immunity, and local mechanism of action within tumor tissues, and discuss further challenges to expediting T-BsAb development.

Rapid, convenient, and highly sensitive detection of human hemoglobin in serum using a high-affinity bivalent antibody-enzyme complex.

Human hemoglobin (Hb) is a biomarker of several diseases, and monitoring of Hb levels is required during emergent surgery. However, rapid and sensitive Hb detection methods are yet to be developed. The present study established a rapid, convenient, and highly sensitive detection method for Hb in human serum using a bivalent antibody-enzyme complex (AEC). AECs are promising sensing elements because of their ability to bind specific targets and their catalytic activity that produce signals. We recently reported a convenient and universal method to fabricate bivalent AECs with two antibody fragments, using the SpyCatcher/SpyTag system. The present study applied a bivalent AEC for highly sensitive and quantitative detection of human Hb. The bivalent anti-Hb AEC was successfully prepared by incubating both N- and C-terminus SpyCatcher-fused glucose dehydrogenase and SpyTag-fused anti-Hb single-chain variable fragments at 4 °C. As expected, the bivalent AEC for Hb with a multimeric structure showed higher affinity than the monovalent AEC, by means of avidity effects, unlike that for soluble epidermal growth factor receptor with a monomeric structure; this contributed to a great improvement in sensitivity. Finally, we established a rapid and wash-free homogeneous electrochemical detection system for Hb by integrating magnetic beads. The linear range of the system completely covered the clinically required Hb levels, even in human serum. This technology provides an ideal point-of-care test for Hb and other multimeric biomarkers.

Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope.

Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR-528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.

Evaluation of intercellular cross-linking abilities correlated with cytotoxicities of bispecific antibodies with domain rearrangements using AFM force-sensing.

Bispecific antibodies (bsAbs) are a promising engineered antibody format; thus, technologies for the fabrication and evaluation of functional bsAbs are attracting increasing attention. Here, based on atomic force microscopy (AFM) force-sensing integrated with a metal cup-attached AFM chip (cup-chip) to ensure efficient capture of a target cell on a cantilever, we established a novel method for measuring cross-linking ability that is correlated with the cytotoxicities of bsAbs targeting two cells. We previously reported that domain rearrangements of bsAbs affected their cytotoxicities; however, no differences in cross-linking ability for soluble antigens were observed by surface plasmon resonance. We predicted that there would be differences in molecular configurations to avoid steric hindrance in the cross-linking of the two whole target cells. A picked-up T cell lymphoma cell on the cantilever using a cup-chip was moved to approach a cancer cell adhered to a dish, and force-curve measurements were performed. The resulting forces mediated by the cross-linking of bsAbs with different domain orders were well-correlated with their cytotoxicities. The AFM force-sensing method established herein may reflect steric hindrance of intercellular cross-linking, and thus has the potential to evaluate the net function of bsAbs and contribute to the generation of functional bsAbs.