Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies.

Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70°C of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.

An immunocytokine consisting of a TNFR2 agonist and TNFR2 scFv enhances the expansion of regulatory T cells through TNFR2 clustering.

Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells.

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

[Functional Study of TNFR2 Signaling and Drug Discovery Using a Protein Engineering Approach].

Tumor necrosis factor-α (TNF), a proinflammatory cytokine, is critical to the pathogenesis of various inflammatory diseases. There are two subtypes of receptors for TNF, namely type I TNF receptor (TNFR1) and type II TNF receptor (TNFR2). Previous studies using animal models of diseases have demonstrated the predominant role of TNFR1 in the pathogenesis of inflammation. It has recently been proposed that TNFR2 is associated with anti-inflammatory function. This intriguing function of TNFR2 has implications from an immunological and pharmacological perspective. However, the mechanism of the TNFR2-mediated anti-inflammatory effect is not fully understood. In this context, we attempted to elucidate the TNFR2-mediated anti-inflammatory effect and other unknown biological functions of TNFR2 by utilizing our protein engineering technology to generate functional mutant cytokines. Our findings reveal the following. (1) TNFR2 is expressed on regulatory T cells (Tregs) but not conventional T cells (Tconvs) and TNFR2-mediated signals promote proliferation and activation of Tregs. (2) The crystal structure of TNF/TNFR2 complex was solved, which suggests a possible signal initiation mechanism via TNF/TNFR2 cluster formation on the cellular membrane. (3) A novel TNFR2-mediated signal molecule, aminopeptidase P3 (APP3/XPNPEP3), was identified that interacts with TNFR2 as an intracellular adaptor protein. APP3 is required for c-Jun N-terminal kinase (JNK) phosphorylation, the downstream molecule of TNFR2 signal transduction. These results are key to understanding the mechanism of immune regulation and will assist in the identification of immunomodulatory drugs targeting the TNFR2 signaling cascade as well as the function of Tregs.

Characterization of a TNFR2-Selective Agonistic TNF-α Mutant and Its Derivatives as an Optimal Regulatory T Cell Expander.

Regulatory T cells (Tregs) are a subpopulation of lymphocytes that play a role in suppressing and regulating immune responses. Recently, it was suggested that controlling the functions and activities of Tregs might be applicable to the treatment of human diseases such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease. TNF receptor type 2 (TNFR2) is a target molecule that modulates Treg functions. In this study, we investigated the role of TNFR2 signaling in the differentiation and activation of mouse Tregs. We previously reported the generation of a TNFR2-selective agonist TNF mutant, termed R2agoTNF, by using our unique cytokine modification method based on phage display. R2agoTNF activates cell signaling via mouse TNFR2. In this study, we evaluated the efficacy of R2agoTNF for the proliferation and activation of Tregs in mice. R2agoTNF expanded and activated mouse CD4+CD25+ Tregs ex vivo. The structural optimization of R2agoTNF by internal cross-linking or IgG-Fc fusion selectively and effectively enhanced Treg expansion in vivo. Furthermore, the IgG-Fc fusion protein suppressed skin-contact hypersensitivity reactions in mice. TNFR2 agonists are expected to be new Treg expanders.

Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics.

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.

A gapmer antisense oligonucleotide targeting SRRM4 is a novel therapeutic medicine for lung cancer.

Small cell lung cancer (SCLC) is the most aggressive neuroendocrine phenotype of the deadliest human lung cancers. However the therapeutic landscape for SCLC has not changed in over 30 years. Effective treatment and prognosis are needed to combat this aggressive cancer. Herein we report that Ser/Arg repetitive matrix 4 (SRRM4), a splicing activator, is abnormally expressed at high levels in SCLC and thus is a potential therapeutic target. We screened an effective gapmer antisense oligonucleotide (gASO) targeting SRRM4 in vitro which led to cell death of SCLC. Our gASO, which is stabilized by containing artificial nucleotides, effectively represses SRRM4 mRNA. We found that our gASO repressed SRRM4 synthesis leading to a dramatic tumor reduction in a lung cancer mouse model. We also analyzed miRNA microarray and found that the miR-4516 is abnormally increased in exosomes in the blood of SCLC patients. Treating with gASO suppressed tumors in the SCLC model mouse concurrently reduced plasma miR-4516. In conclusion this study reports that administration of an SRRM4-targeted gASO coupled with a novel miRNA diagnostic methodology represents a potential breakthrough in the therapeutic treatment of high mortality SCLC.

Neutrophil Depletion Exacerbates Pregnancy Complications, Including Placental Damage, Induced by Silica Nanoparticles in Mice.

Recent advances in nanotechnology have led to the development of nanoparticles with innovative functions in various fields. However, the biological effects of nanoparticles-particularly those on the fetus-need to be investigated in detail, because several previous studies have shown that various nanoparticles induce pregnancy complications in mice. In this regard, our previous findings in mice suggested that the increase in peripheral neutrophil count induced by treatment with silica nanoparticles with a diameter of 70 nm (nSP70) may play a role in the associated pregnancy complications. Therefore, here, we sought to define the role of neutrophils in nSP70-induced pregnancy complications. The peripheral neutrophil count in pregnant BALB/c mice at 24 h after treatment with nSP70 was significantly higher than in saline-treated mice. In addition, maternal body weight, uterine weight, and the number of fetuses in nSP70-treated mice pretreated with anti-antibodies, which deplete neutrophils, were significantly lower than those in nSP70-treated mice pretreated with phosphate-buffered saline or isotype-matched control antibodies. Histology revealed that neutrophil depletion increased nSP70-induced placental damage from the decidua through the spongiotrophoblast layer and narrowed spiral arteries in the placentae. In addition, depletion of neutrophils augmented nSP70-induced cytotoxicity to fetal vessels, which were covered with endothelium. The rate of apoptotic cell death was significantly higher in the placentae of anti-nSP70-treated mice than in those from mice pretreated with isotype-matched control antibodies. Therefore, impairment of placental vessels and apoptotic cell death due to nSP70 exposure is exacerbated in the placentae of nSP70-treated mice pretreated with anti-antibodies. Depletion of neutrophils worsens nSP70-induced pregnancy complications in mice; this exacerbation was due to enhanced impairment of placental vessels and increased apoptotic cell death in maternal placentae. Our results provide basic information regarding the mechanism underlying silica-nanoparticle-induced pregnancy complications.

[Development of a Fundamental Technology to Seek Drug Targets, and Its Application to Cancer Targeting Therapy].

　Human epidermal growth factor receptor 2 (Her2)-targeting antibodies and anti-hormone therapy are effective for most breast cancer patients. However, such approaches are not viable with resistant cases or in triple-negative breast cancer (TNBC) patients, given the lack of Her2 and estrogen and progesterone receptors in these patients. Thus, new drug targets are urgently required. From this perspective, we searched for novel drug targets using proteomic analysis, and identified Eph receptor A10 (EphA10), which is elevated in breast cancer cells as compared to normal breast tissue. Here, we evaluated the potential of EphA10 as a drug target by analyzing its protein expression profile/function in cancer cells, and then by using an anti-EphA10 antibody to treat EphA10-expressing tumor-bearing mice. Protein expression profile analysis showed that EphA10 was expressed in various breast cancer subtypes, including TNBCs, with no expression observed in normal tissues, apart from the testes. Moreover, functional analysis of the cancer cells revealed that ligand-dependent proliferation was observed in EphA10-expressed cancer cells. Thus, we developed our novel anti-EphA10 antibody, which binds to EphA10 with high specificity and affinity at the nanomolar level. Finally, therapeutic analysis indicated that tumor growth was significantly suppressed in the mAb-treated mice in a dose-dependent manner. These results suggest that the EphA10-targeting therapy may be a novel therapeutic option for the management of breast cancer, including in TNBCs which aren't currently treated with molecular-targeted agents. Consequently, we hope that these findings will contribute to the development of a new targeting therapy for refractory breast cancer patients.

Modifying the Surface of Silica Nanoparticles with Amino or Carboxyl Groups Decreases Their Cytotoxicity to Parenchymal Hepatocytes.

We previously reported that unmodified silica nanoparticles with diameters of 70 nm (nSP70) induced liver damage in mice, whereas nSP70 modified with carboxyl or amino groups did not. In addition, we have found that both unmodified and modified nSP70s localize in both Kupffer cells and parenchymal hepatocytes. We therefore evaluated the contributions of nSP70 uptake by these cell populations to liver damage. To this end, we pretreated mice with gadolinium (III) chloride hydrate (GdCl3) to prevent nSP70 uptake by Kupffer cells, subsequently injected the mice with either type of nSP70, and then assessed plasma levels of alanine aminotransferase (ALT). In mice given GdCl3, unmodified nSP70 increased ALT levels. From these data, we hypothesized that in GdCl3-treated mice, the unmodified nSP70 that was prevented from entering Kupffer cells was shunted to parenchymal hepatocytes, where it induced cytotoxicity and increased liver damage. In contrast, GdCl3 pretreatment had no effect on ALT levels in mice injected with surface-modified nSP70s, suggesting that modified nSP70s spared parenchymal hepatocytes and thus induced negligible liver damage. In cytotoxicity analyses, the viability of a parenchymal hepatocyte line was greater when exposed to surface-modified nSP70s than to unmodified nSP70s. These findings imply that the decreased liver damage associated with surface-modified compared with unmodified nSP70 is attributable to decreased cytotoxicity to parenchymal hepatocytes.

Identifying a size-specific hazard of silica nanoparticles after intravenous administration and its relationship to the other hazards that have negative correlations with the particle size in mice.

Many of the beneficial and toxic biological effects of nanoparticles have been shown to have a negative correlation with particle size. However, few studies have demonstrated biological effects that only occur at specific nanoparticle sizes. Further elucidation of the size-specific biological effects of nanoparticles may reveal not only unknown toxicities, but also novel benefits of nanoparticles. We used surface-unmodified silica particles with a wide range of diameters and narrow size intervals between the diameters (10, 30, 50, 70, 100, 300, and 1000 nm) to investigate the relationship between particle size and acute toxicity after intravenous administration in mice. Negative correlations between particle size and thrombocytopenia, liver damage, and lethal toxicity were observed. However, a specific size-effect was observed for the severity of hypothermia, where silica nanoparticles with a diameter of 50 nm induced the most severe hypothermia. Further investigation revealed that this hypothermia was mediated not by histamine, but by platelet-activating factor, and it was independent of the thrombocytopenia and the liver damage. In addition, macrophages/Kupffer cells and platelets, but not neutrophils, play a critical role in the hypothermia. The present results reveal that silica nanoparticles have particle size-specific toxicity in mice, suggesting that other types of nanoparticles may also have biological effects that only manifest at specific particle sizes. Further study of the size-specific effects of nanoparticles is essential for safer and more effective nanomedicines.

A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification.

Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.

Distribution of Silver Nanoparticles to Breast Milk and Their Biological Effects on Breast-Fed Offspring Mice.

Recent rodent studies have shown that nanoparticles are distributed to breast milk, and more-detailed safety information regarding nanoparticle consumption by lactating mothers is required. Here, we used mice to investigate the safety of nanoparticle use during lactation. When Ag and Au nanoparticles were intravenously administered to lactating mice, the nanoparticles were distributed to breast milk without producing apparent damage to the mammary gland, and the amount of Ag nanoparticles distributed to breast milk increased with decreasing particle size. Orally administered Ag nanoparticles were also distributed to breast milk and subsequently to the brains of breast-fed pups. Ten-nanometer Ag nanoparticles were retained longer in the pups' brains than in their livers and lungs. Nevertheless, no significant behavioral changes were observed in offspring breast-fed by dams that had received orally administered 10 nm Ag nanoparticles. These data provide basic information for evaluating the safety of nanoparticle use during lactation.

Moesin is activated in cardiomyocytes in experimental autoimmune myocarditis and mediates cytoskeletal reorganization with protrusion formation.

Acute myocarditis is a self-limiting disease. Most patients with myocarditis recover without cardiac dysfunction in spite of limited capacity of myocardial regeneration. Therefore, to address intrinsic reparative machinery of inflamed hearts, we investigated the cellular dynamics of cardiomyocytes in response to inflammation using experimental autoimmune myocarditis (EAM) model. EAM was induced by immunization of BALB/c mice with α-myosin heavy chain peptides twice. The inflammatory reaction was evoked with myocardial damage with the peak at 3 wk after the first immunization (EAM3w). Morphological and functional restoration started from EAM3w, when active protrusion formation, a critical process of myocardial healing, was observed in cardiomyocytes. Shotgun proteomics revealed that cytoskeletal proteins were preferentially increased in cardiomyocytes at EAM3w, compared with preimmunized (EAM0w) hearts, and that moesin was the most remarkably upregulated among them. Immunoblot analyses demonstrated that the expression of both total and phosphorylated moesin was upregulated in isolated cardiomyocytes from EAM3w hearts. Immunofluorescence staining showed that moesin was localized at cardiomyocyte protrusions at EAM3w. Adenoviral vectors expressing wild-type, constitutively active and inactive form of moesin (wtMoesin, caMoesin, and iaMoesin, respectively) were transfected in neonatal rat cardiomyocytes. The overexpression of wtMoesin and caMoesin resulted in protrusion formation, while not iaMoesin. Finally, we found that cardiomyocyte protrusions were accompanied by cell-cell contact formation. The expression of moesin was upregulated in cardiomyocytes under inflammation, inducing protrusion formation in a phosphorylation-dependent fashion. Moesin signal could be a novel therapeutic target that stimulates myocardial repair by promoting contact formation of cardiomyocytes.

Metal nanoparticles in the presence of lipopolysaccharides trigger the onset of metal allergy in mice.

Many people suffer from metal allergy, and the recently demonstrated presence of naturally occurring metal nanoparticles in our environment could present a new candidate for inducing metal allergy. Here, we show that mice pretreated with silver nanoparticles (nAg) and lipopolysaccharides, but not with the silver ions that are thought to cause allergies, developed allergic inflammation in response to the silver. nAg-induced acquired immune responses depended on CD4(+) T cells and elicited IL-17A-mediated inflammation, similar to that observed in human metal allergy. Nickel nanoparticles also caused sensitization in the mice, whereas gold and silica nanoparticles, which are minimally ionizable, did not. Quantitative analysis of the silver distribution suggested that small nAg (≤10 nm) transferred to the draining lymph node and released ions more readily than large nAg (>10 nm). These results suggest that metal nanoparticles served as ion carriers to enable metal sensitization. Our data demonstrate a potentially new trigger for metal allergy.

The Matrix Proteins Hasp and Hig Exhibit Segregated Distribution within Synaptic Clefts and Play Distinct Roles in Synaptogenesis.

The synaptic cleft is the space through which neurotransmitters convey neural information between two synaptic terminals. This space is presumably filled with extracellular matrix molecules involved in synaptic function or differentiation. However, little is known about the identities of the matrix components, and it remains unclear how these molecules organize the matrix in synaptic clefts. In this study, we identified Hasp, a Drosophila secretory protein containing CCP and WAP domains. Molecular genetic analysis revealed that Hasp diffuses extracellularly and is predominantly captured at synaptic clefts of cholinergic synapses. Furthermore, Hasp regulates levels of DLG and the nAChR subunits Dα6 and Dα7 at postsynaptic terminals. Hasp is required for trapping of another matrix protein, Hig, which is also secreted and diffused in the brain, at synaptic clefts of cholinergic synapses; however, Hig is dispensable for localization of Hasp at synaptic clefts. In addition, in the brains of triple mutants for the nAChR subunits Dα5, Dα6, and Dα7, the level of Hig, but not Hasp, was markedly reduced in synaptic regions, indicating that these nAChR subunits are required to anchor Hig to synaptic clefts. High-resolution microscopy revealed that Hasp and Hig exhibit segregated distribution within individual synaptic clefts, reflecting their differing roles in synaptogenesis. These data provide insight into how Hasp and Hig construct the synaptic cleft matrix and regulate the differentiation of cholinergic synapses, and also illuminate a previously unidentified architecture within synaptic clefts. SIGNIFICANCE STATEMENT: The synapse has been extensively studied because it is essential for neurotransmission. By contrast, the space between the synaptic terminals, the synaptic cleft, is still an undeveloped research area despite its ubiquity in synapses. In fruit fly brains, we obtained evidence that the matrix protein Hasp and the previously identified Hig, both of which are secreted extracellularly, localize predominantly to synaptic clefts of cholinergic synapses, and modulate the levels of nAChR subunits on postsynaptic membranes. However, Hasp and Hig play differential roles in matrix formation and exhibit segregated distribution within synaptic clefts. These results reveal the molecular mechanisms of synaptic matrix construction and illuminate a molecular architecture within synaptic clefts previously unrevealed in any animal species.

Consecutive incorporation of functionalized nucleotides with amphiphilic side chains by novel KOD polymerase mutant.

Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.

Furospinosulin-1, Marine Spongean Furanosesterterpene, Suppresses the Growth of Hypoxia-Adapted Cancer Cells by Binding to Transcriptional Regulators p54(nrb) and LEDGF/p75.

Hypoxia-adapted cancer cells in tumors contribute to the pathological progression of cancer. Cancer research has therefore focused on the identification of molecules responsible for hypoxia adaptation in cancer cells, as well as the development of new compounds with action against hypoxia-adapted cancer cells. The marine natural product furospinosulin-1 (1) has displayed hypoxia-selective growth inhibition against cultured cancer cells, and has shown in vivo anti-tumor activity, although its precise mode of action and molecular targets remain unclear. In this study, we found that 1 is selectively effective against hypoxic regions of tumors, and that it directly binds to the transcriptional regulators p54(nrb) and LEDGF/p75, which have not been previously identified as mediators of hypoxia adaptation in cancer cells.

Creation of mouse TNFR2-selective agonistic TNF mutants using a phage display technique.

Tumor necrosis factor-α (TNF), which is an immuno-modulatory cytokine, has been suggested to cause inflammatory responses as well as protection against tissue dysfunction by binding two types of TNF receptor (TNFR1/TNFR2). However, the physiological effects of TNFR2-specific activation remain unclear. We therefore aimed to generate a TNF mutant with full TNFR2-selective agonist activity as a functional analytical tool. In this study, we utilized a phage display technique to create mouse TNFR2 (mTNFR2)-selective TNF mutants that bind specifically to mTNFR2 and show full bioactivity compared with wild-type TNF. A new phage library displaying TNF mutants was created, in which nine amino acid residues at the predicted receptor-binding site were randomized. From this library, an agonistic TNF mutant exhibiting high binding selectivity and bioactivity to mTNFR2 was isolated. We propose that this TNF mutant would be a powerful tool with which to elucidate the functional roles of mTNFR2.