Development of DNA aptamers universally bound to single-chain fragment variables and their applications in bioprocess monitoring.

Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with KD = 2.1 µM or KD = 1.6 µM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

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.

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.

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.

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.

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.

Development of glycated peptide enzyme sensor based flow injection analysis system for haemoglobin A1c monitoring using quasi-direct electron transfer type engineered fructosyl peptide oxidase.

Haemoglobin A1c (hemoglobin A1c, HbA1c) is an important long-term glycemic control marker for diabetes. The aim of this study was to develop an enzyme flow injection analysis (FIA) system using engineered fructosyl peptide oxidase (FPOx) based on 2.5th generation principle for an HbA1c automated analytical system. FPOx from Phaeosphaeria nodorum (PnFPOx) was engineered by introducing a Lys residue at the R414 position, to be modified with amine reactive phenazine ethosulfate (arPES) in proximity of FAD. The engineered PnFPOx mutant with minimized oxidase activity, N56A/R414K, showed quasi-direct electron transfer (quasi-DET) ability after PES-modification. The FIA system was constructed by employing a PES-modified PnFPOx N56A/R414K and operated at 0 V against Ag/AgCl. The system showed reproducible responses with a linear range of 20-500 µM for both fructosyl valine (FV) and fructosyl valylhistidine (FVH), with sensitivities of 0.49 nA µM-1 and 0.13 nA µM-1, and the detection limits of 1.3 µM and 2.0 µM for FV and FVH, respectively. These results indicate that the enzyme electrochemical FIA system covers the clinical range of HbA1c detection for more 200 consecutive measurements. Protease digested three different levels of HbA1c samples including healthy and diabetic range subjects were also measured with the FIA system. Thus, it will be possible to develop an integrated system consisting of sample pretreatment and sample electrochemical measurement based on an FIA system possessing quasi-DET type PnFPOx.

Rapid and homogeneous electrochemical detection by fabricating a high affinity bispecific antibody-enzyme complex using two Catcher/Tag systems.

Antibody-enzyme complexes (AECs) with binding ability to specific targets and catalytic activities to gain signals are known to be ideal sensing elements; however, AEC-based universal sensors applicable to point-of-care testing (POCT) have not yet been developed. Here, we achieved rapid and homogeneous electrochemical detection by fabricating a high-affinity bispecific AEC (bsAEC) using two Catcher/Tag systems. Recently, we reported a convenient and universal method to fabricate AECs using the SpyCatcher/SpyTag system. The resultant anti-epidermal growth factor receptor (anti-EGFR) AEC worked efficiently as a sensing element; however, the sensitivities did not meet the clinically required detection range of the soluble ectodomain of EGFR (sEGFR). To induce high affinity even to monomeric targets like sEGFR, we designed a convenient fabrication method for bsAEC using two Catcher/Tag systems, which did not express cross-reactivity. The anti-EGFR bsAEC was successfully prepared by constructing glucose dehydrogenase with two different catcher domains at the N- and C-terminus and by combining two corresponding Tag-fused anti-EGFR single-chain Fvs (scFvs), which recognize different epitopes on sEGFR. As expected, bsAEC showed a higher affinity than that of bivalent AEC with two identical anti-EGFR scFvs at low concentrations of sEGFR, and met the clinically required detection range of sEGFR. Further, by combining magnet beads, we established a rapid and wash-free homogeneous electrochemical detection method. This study offers new insights into the fabrication of universal POCT devices.

Mechanism of action of a T cell-dependent bispecific antibody as a breakthrough immunotherapy against refractory colorectal cancer with an oncogenic mutation.

T cell-dependent bispecific antibody (TDB)-induced T cell activation, which can eliminate tumor cells independent of MHC engagement, is expected to be a novel breakthrough immunotherapy against refractory cancer. However, the mechanism of action of TDBs has not been fully elucidated thus far. We focused on TDB-induced T cell-tumor cell contact as an important initial step in direct T cell-mediated tumor cell killing via transport of cytotoxic cell proteases (e.g., granzymes) with or without immunological synapse formation. Using an anti-EGFR/CD3 TDB, hEx3, we visualized and quantified T cell-tumor cell contact and demonstrated a correlation between the degree of cell contact and TDB efficacy. We also found that cytokines, including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) secreted by activated T cells, damaged tumor cells in a cell contact-independent manner. Moreover, therapeutic experiences clearly indicated that hEx3, unlike conventional anti-EGFR antibodies, was effective against colorectal cancer (CRC) cells with mutant KRAS, BRAF, or PIK3CA. In a pharmacokinetic analysis, T cells spread gradually in accordance with the hEx3 distribution within tumor tissue. Accordingly, we propose that TDBs should have four action steps: 1st, passive targeting via size-dependent tumor accumulation; 2nd, active targeting via specific binding to tumor cells; 3rd, T cell redirection toward tumor cells; and 4th, TDB-induced cell contact-dependent (direct) or -independent (indirect) tumor cell killing. Finally, our TDB hEx3 may be a promising reagent against refractory CRC with an oncogenic mutation associated with a poor prognosis.

Functional Domain Order of an Anti-EGFR × Anti-CD16 Bispecific Diabody Involving NK Cell Activation.

Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.

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.

Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues-which can be chemical conjugation sites-were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites.

A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.

Convenient and Universal Fabrication Method for Antibody-Enzyme Complexes as Sensing Elements Using the SpyCatcher/SpyTag System.

Antibody-enzyme complexes (AECs) are ideal sensing elements, especially when oxidoreductases are used as the enzymes in the complex, with the potential to carry out rapid electrochemical measurements. However, conventional methods for the fabrication of AECs, including direct fusion and chemical conjugation, are associated with issues regarding the generation of insoluble aggregates and production of homogeneous AECs. Here, we developed a convenient and universal method for the fabrication of homogeneous AECs using the SpyCatcher/SpyTag system. We used an anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain antibody (VHH) and a glucose dehydrogenase (GDH) derived from Aspergillus flavus ( AfGDH) as the model antibody and enzyme, respectively. Both SpyTag-fused VHH and SpyCatcher-fused AfGDH were successfully prepared using an Escherichia coli expression system, whereas anti-EGFR AECs were produced by simply mixing the two fusion proteins. A bivalent AEC, AfGDH with two VHH at both terminals, was also prepared and exhibited an increased affinity. A soluble EGFR was successfully detected in a dose-dependent manner using immobilized anti-EGFR immunoglobulin G (IgG) and bivalent AEC. We also confirmed the universality of this AEC fabricating method by applying it to another VHH. This method results in the convenient and universal preparation of sensing elements with the potential for electrochemical measurement.

Engineering the hinge region of human IgG1 Fc-fused bispecific antibodies to improve fragmentation resistance.

Fc domain fusion can improve the therapeutic effects of relatively small biological molecules such as peptides, cytokines, and antibody fragments. Fc fusion proteins can also be used to enhance the cytotoxic effects of small bispecific antibodies (bsAbs). However, fragmentation of Fc fusion proteins, which mainly occurs around the hinge regions during production, storage, and circulation in the blood, is a major issue. In this study, we first investigated the mechanisms of fragmentation around the hinge region during storage using Fc-fused bsAbs with specificity for epidermal growth factor receptor and CD3 as a model. The fragmentation peaks generated by gel filtration analysis indicated that both contaminating proteases and dissolved active oxygen should be considered causes of fragmentation. We designed and constructed variants by introducing a point mutation into the upper hinge region, which reduced the cleavage caused by dissolved active oxygen, and shortened the hinge region to restrict access of proteases. These hinge modifications improved fragmentation resistance and did not affect the biological activity of the bsAbs in vitro. We confirmed the versatility of the hinge modifications using another Fc-fused bsAb. Our results show that hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially, and these modifications can be used to improve the fragmentation resistance of other recombinant Fc fusion proteins.

Esterification of PQQ Enhances Blood-Brain Barrier Permeability and Inhibitory Activity against Amyloidogenic Protein Fibril Formation.

Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of α-synuclein, amyloid ß1-42 (Aß1-42), and mouse prion protein. To develop a novel drug against neurodegenerative diseases based on PQQ, it is necessary to improve the insufficient BBB permeability of PQQ. Here, we show that an esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), has twice the BBB permeability than PQQ in vitro. Moreover, PQQ-TME exhibited greater inhibitory activity against fibrillation of α-synuclein, Aß1-42, and prion protein. These results indicated that esterification of PQQ could be a useful approach in developing a novel PQQ-based amyloid inhibitor.