Humanization and characterization of an anti-ciguatoxin CTX3C monoclonal antibody.

Ciguatera poisoning (CP), caused by ciguatoxins (CTXs), is one of the most common food-borne diseases, affecting more than 50,000 people each year. In most cases, CP are managed with symptomatic and supportive remedies, and no specific treatment has been devised. In this study, toward the development of therapeutic antibodies for CP, we examined to humanize mouse anti-CTX3C antibody 10C9 (m10C9), which exhibited neutralizing activity against ciguatoxin in vitro and in vivo. The complementarity determining regions were grafted onto a human germline sequence with high sequence identity to m10C9, and the backmutations were examined to maintain the binding affinity. The optimized humanized antibody, Opt.h10C9Fab, showed a strong binding affinity to CTX3C with a high affinity (KD = 19.0 nM), and only two backmutations of ArgL46 and CysH94 in the framework regions were involved in determining the antigen binding affinity.

Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

Inkjet-Based Intracellular Delivery System that Effectively Utilizes Cell-Penetrating Peptides for Cytosolic Introduction of Biomacromolecules through the Cell Membrane.

In the drug delivery system, the cytosolic delivery of biofunctional molecules such as enzymes and genes must achieve sophisticated activities in cells, and microinjection and electroporation systems are typically used as experimental techniques. These methods are highly reliable, and they have high intracellular transduction efficacy. However, a high degree of proficiency is necessary, and induced cytotoxicity is considered as a technical problem. In this research, a new intracellular introduction technology was developed through the cell membrane using an inkjet device and cell-penetrating peptides (CPPs). Using the inkjet system, the droplet volume, droplet velocity, and dropping position can be accurately controlled, and minute samples (up to 30 pL/shot) can be carried out by direct administration. In addition, CPPs, which have excellent cell membrane penetration functions, can deliver high-molecular-weight drugs and nanoparticles that are difficult to penetrate through the cell membrane. By using the inkjet system, the CPPs with biofunctional cargo, including peptides, proteins such as antibodies, and exosomes, could be accurately delivered to cells, and efficient cytosolic transduction was confirmed.

T Cell-Association of Carboxy-Terminal Dendrimers with Different Bound Numbers of Phenylalanine and Their Application to Drug Delivery.

T cells play important roles in various immune reactions, and their activation is necessary for cancer immunotherapy. Previously, we showed that polyamidoamine (PAMAM) dendrimers modified with 1,2-cyclohexanedicarboxylic acid (CHex) and phenylalanine (Phe) underwent effective uptake by various immune cells, including T cells and their subsets. In this study, we synthesized various carboxy-terminal dendrimers modified with different bound numbers of Phe and investigated the association of these dendrimers with T cells to evaluate the influence of terminal Phe density. Carboxy-terminal dendrimers conjugating Phe at more than half of the termini exhibited a higher association with T cells and other immune cells. The carboxy-terminal Phe-modified dendrimers at 75% Phe density tended to exhibit the highest association with T cells and other immune cells, which was related to their association with liposomes. A model drug, protoporphyrin IX (PpIX), was encapsulated into carboxy-terminal Phe-modified dendrimers, which were then used for drug delivery into T cells. Our results suggest the carboxy-terminal Phe-modified dendrimers are useful for delivery into T cells.

Generation of molecular-targeting helix-loop-helix peptides for inhibition of the interaction between cytotoxic T-lymphocyte-associated protein 4 and B7 in the dog.

Blocking the interaction between CD28 and B7 by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a potent immune checkpoint that prevents damage to host tissues from excessive immune responses. However, it also significantly diminishes immune responses against cancers and allows cancer cell growth. This study found that recombinant (r) human (h) CTLA-4 specifically binds to canine dendritic cells (DCs) and suppresses the responses of canine T cells to allogeneic DCs. ERY2-4, a peptide targeting rhCTLA-4 selected from a yeast-displayed library of helix-loop-helix (HLH) peptides and improved to have a binding affinity to rhCTLA-4 as strong as that of rhB7, inhibited the binding of rhCTLA-4 to canine DCs. Furthermore, the targeting peptide significantly enhanced the response of canine T cells to allogeneic DCs. These results suggest that the CTLA-4-targeting peptide enhances canine T cell activity by blocking the interaction between canine CTLA-4 on T cells and canine B7 on DCs. This study demonstrates the generation of a new type of immune checkpoint inhibitor, which may be applicable to cancer therapy in dogs.

"Human and Mouse Cross-Reactive" Albumin-Binding Helix-Loop-Helix Peptide Tag for Prolonged Bioactivity of Therapeutic Proteins.

The effectiveness of protein and peptide pharmaceuticals depends essentially on their intrinsic pharmacokinetics. Small-sized pharmaceuticals in particular often suffer from short serum half-lives due to rapid renal clearance. To improve the pharmacokinetics by association with serum albumin (SA) in vivo, we generated an SA-binding tag of a helix-loop-helix (HLH) peptide to be linked with protein pharmaceuticals. For use in future preclinical studies, screening of yeast-displayed HLH peptide libraries against human SA (HSA) and mouse SA (MSA) was alternately repeated to give the SA-binding peptide AY-VE, which exhibited cross-binding activities to HSA and MSA with KD of 65 and 20 nM, respectively. As a proof of concept, we site-specifically conjugated peptide AY-VE with insulin to examine its bioactivity in vivo. In mouse bioassay monitoring the blood glucose level, the AY-VE conjugate was found to have a prolonged hypoglycemic effect for 12 h. The HLH peptide tag is a general platform for extending the bioactivity of therapeutic peptides or proteins.

Dodecaborate-Encapsulated Extracellular Vesicles with Modification of Cell-Penetrating Peptides for Enhancing Macropinocytotic Cellular Uptake and Biological Activity in Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a radiation therapy for cancer. In BNCT, the internalization of boron-10 atoms by cancer cells induces cell death through the generation of α particles and recoiling lithium-7 nuclei when irradiated with low-energy thermal neutrons. In this study, we aimed to construct exosomes [extracellular vesicles (EVs)]-based drug delivery technology in BNCT. Because of their pharmaceutical advantages, such as controlled immune responses and effective usage of cell-to-cell communication, EVs are potential next-generation drug delivery carriers. In this study, we successfully developed polyhedral borane anion-encapsulated EVs with modification of hexadeca oligoarginine, which is a cell-penetrating peptide, on the EV membrane to induce the actin-dependent endocytosis pathway, macropinocytosis, which leads to efficient cellular uptake and remarkable cancer cell-killing BNCT activity. The simple and innovative technology of the EV-based delivery system with "cassette" modification of functional peptides will be applicable not only for BNCT but also for a wide variety of therapeutic methodologies.

New Class of Drug Modalities: Directed Evolution of a De Novo Designed Helix-Loop-Helix Peptide to Bind VEGF for Tumor Growth Inhibition.

As a small affinity molecule to serve as an alternative to antibodies, we have developed a conformationally constrained peptide with a de novo designed helix-loop-helix (HLH) scaffold. To evaluate its potential for biomedical applications, we performed directed evolution of HLH peptides to obtain an inhibitor for vascular endothelial growth factor-A (VEGF). A phage-displayed library of HLH peptides was constructed and screened against VEGF, giving the peptide VS42 that inhibits the VEGF/VEGF receptor-2 interaction (IC50 = 210 nM), which was further improved by in vitro affinity maturation using a yeast-displayed library. An identified HLH peptide, VS42-LR3, exhibited improved inhibitory activity (IC50 = 37 nM), high thermal stability, and excellent resistance against chemical denaturation. In biological activity tests, the HLH peptide was found to block VEGF-induced proliferation of human umbilical vein endothelial cells and suppress tumor growth in a murine xenograft model of human colorectal cancer.

Carboxy-terminal dendrimers with phenylalanine for a pH-sensitive delivery system into immune cells including T cells.

Although T cells play important roles in various immune reactions, there are only a few reports on delivery systems into T cells. Our previous study showed that carboxy-terminal phenylalanine (Phe)-modified polyamidoamine (PAMAM) dendrimers have both temperature- and pH-sensitive properties, which are affected by the chemical structure. The self-assembled structures of Phe, observed in phenylketonuria, enhance the protein aggregation, the association with the cell membrane and the membrane permeability. In this study, we applied the Phe-modified dendrimers to a pH-sensitive drug delivery system into T cells. Dendrimers with different amino acids and acid anhydrides were synthesized, and their pH-responsive association with T cells and their subsets was investigated. The dendrimers modified with Phe and cyclohexanedicarboxylic acid (CHex) showed higher uptake into various cells, including Jurkat cells, CD3+ T cells, CD3 + CD4+ helper T cells and CD3 + CD8+ killer T cells. These dendrimers were internalized into T cells via endocytosis, and their cellular uptake was enhanced under weak acidic conditions (pH 6.5). Our results showed that Phe- and CHex-modified dendrimers have a delivery potential to T cells and their subsets, which may be useful for cancer immunotherapy.

Structure of mitogen-activated protein kinase kinase 1 in the DFG-out conformation.

Eukaryotic protein kinases contain an Asp-Phe-Gly (DFG) motif, the conformation of which is involved in controlling the catalytic activity, at the N-terminus of the activation segment. The motif can be switched between active-state (DFG-in) and inactive-state (DFG-out) conformations: however, the mechanism of conformational change is poorly understood, partly because there are few reports of the DFG-out conformation. Here, a novel crystal structure of nonphosphorylated human mitogen-activated protein kinase kinase 1 (MEK1; amino acids 38-381) complexed with ATP-γS is reported in which MEK1 adopts the DFG-out conformation. The crystal structure revealed that the structural elements (the αC helix and HRD motif) surrounding the active site are involved in the formation/stabilization of the DFG-out conformation. The ATP-γS molecule was bound to the canonical ATP-binding site in a different binding mode that has never been found in previously determined crystal structures of MEK1. This novel ATP-γS binding mode provides a starting point for the design of high-affinity inhibitors of nonphosphorylated inactive MEK1 that adopts the DFG-out conformation.

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries.

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip-3 retained the α-helical structure and bound to AurA with a KD value of 13.7 µM. Bip-3 and the adenosine-tethered peptide Bip-3-Adc provided IC50 values of 103 µM and 7.7 µM, respectively, suggesting that Bip-3-Adc bivalently inhibited AurA. In addition, the selectivity of Bip-3-Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.

Macropinocytosis-Inducible Extracellular Vesicles Modified with Antimicrobial Protein CAP18-Derived Cell-Penetrating Peptides for Efficient Intracellular Delivery.

The antimicrobial protein CAP18 (approximate molecular weight: 18 000), which was first isolated from rabbit granulocytes, comprises a C-terminal fragment that has negatively charged lipopolysaccharide binding activity. In this study, we found that CAP18 (106-121)-derived (sC18)2 peptides have macropinocytosis-inducible biological functions. In addition, we found that these peptides are highly applicable for use as extracellular vesicle (exosomes, EV)-based intracellular delivery, which is expected to be a next-generation drug delivery carrier. Here, we demonstrate that dimerized (sC18)2 peptides can be easily introduced on EV membranes when modified with a hydrophobic moiety, and that they show high potential for enhanced cellular uptake of EVs. By glycosaminoglycan-dependent induction of macropinocytosis, cellular EV uptake in targeted cells was strongly increased by the peptide modification made to EVs, and intriguingly, our herein presented technique is efficiently applicable for the cytosolic delivery of the biologically cell-killing functional toxin protein, saporin, which was artificially encapsulated in the EVs by electroporation, suggesting a useful technique for EV-based intracellular delivery of biofunctional molecules.

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.

A "ligand-targeting" peptide-drug conjugate: Targeted intracellular drug delivery by VEGF-binding helix-loop-helix peptides via receptor-mediated endocytosis.

As a new alternative to antibody-drug conjugates, we generated "ligand-targeting" peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.

An Immune-Stimulatory Helix-Loop-Helix Peptide: Selective Inhibition of CTLA-4-B7 Interaction.

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC50 of 1.1 ± 0.03 µM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.

Intracellular target delivery of cell-penetrating peptide-conjugated dodecaborate for boron neutron capture therapy (BNCT).

In this study, we designed and synthesized organelle-targeted cell-penetrating peptide (CPP)-conjugated boron compounds to increase their cellular uptake and to control the intracellular locations for the induction of sophisticated anticancer biological activity in boron neutron capture therapy (BNCT), leading to anticancer effects with ATP reduction and apoptosis when irradiated with neutrons in an in vitro BNCT assay.

Highly Sensitive and Practical Fluorescent Sandwich ELISA for Ciguatoxins.

Ciguatera fish poisoning (CFP) caused by the consumption of fish that have accumulated ciguatoxins (CTXs) affects more than 50000 people annually. The spread of CFP causes enormous damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions. The difficulty in avoiding CFP arises from the lack of sensitive and reliable analytical methods for the detection and quantification of CTXs in contaminated fish, along with the normal appearance, smell, and taste of fish contaminated with the causative toxins. Thus, an accurate, sensitive, routine, and portable detection method for CTXs is urgently required. We have successfully developed a highly sensitive fluorescent sandwich ELISA, which can detect, differentiate, and quantify four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit of less than 1 pg/mL. The ELISA protocol, using one microtiter plate coated with two mAbs (10C9 and 3G8), and ALP-linked 8H4, can detect any of the four CTX congeners in a single operation. CTX1B spiked into fish at the FDA guidance level of 0.01 ppb CTX1B equivalent toxicity in fish from Pacific regions was also proven to be reliably detected by this ELISA. Furthermore, the efficiency of extraction/purification procedures and the matrix effect of contaminants in fish were evaluated in detail, since pretreatment and matrix effects are critical for ELISA analysis.

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters.

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (AsnH33, SerH95, and ArgL96) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnH33Ala, SerH95Ala, and ArgL96Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry.