PEG-lipid-modified agonistic antibody against tumor necrosis factor receptor family elicits superior apoptosis-inducing activity against human carcinoma.

We have recently developed a novel PEG-lipid-modified antibody to enhance the induction of apoptosis by the agonistic antibody. The chemically modified TRA-8 antibody [anti-death receptor 5 (DR5) antibody] with PEG-lipid (DSPE-PEG) demonstrated significant cytotoxic activity in vitro without the need for crosslinking with a secondary antibody, which is typically required. We investigated the correlation between the PEG-lipid structure and the cytotoxic activity of the modified antibodies by varying the PEG length or lipid structure. However, when the DSPE-PEG-modified TRA-8 antibody was incubated with plasma, it lost its cytotoxic activity, likely due to degradation in the DSPE-PEG component. Nevertheless, by designing new PEG-lipids that are intended to be resistant to enzymatic degradation, we were able to prevent this degradation and restore the cytotoxic activity of the modified antibody. These findings provide valuable insights for the design of PEG-lipid-modified antibodies and suggest their potential effectiveness in enhancing cancer therapy.

Novel Immunoliposome Technology for Enhancing the Activity of the Agonistic Antibody against the Tumor Necrosis Factor Receptor Superfamily.

We have developed a technology for efficiently enhancing the anticancer apoptosis-inducing activity of agonistic antibodies against the tumor necrosis factor receptor (TNFR) superfamily by the formation of immunoliposomes. To induce apoptosis in cancer cells, agonistic antibodies to the TNFR superfamily normally need cross-linking by internal immune effector cells via the Fc region after binding to receptors on the cell membrane. To develop apoptosis-inducing antibodies that do not require the support of cross-linking by immune cells, we prepared immunoliposomes conjugated with TRA-8, an agonistic antibody against death receptor 5 (DR5), with various densities of antibody on the liposome surface, and evaluated their activities. The TRA-8 immunoliposomes exhibited apoptosis-inducing activity against various DR5-positive human carcinoma cells at a significantly lower concentration without cross-linking than that of the original TRA-8 and its natural ligand (TRAIL). The activity of the immunoliposomes was correlated with the density of antibodies on the surface. As the antibody component, not only the full-length antibody but also the Fab' fragment could be used, and the TRA-8 Fab' immunoliposomes also showed exceedingly high activity compared with the parental antibody, namely, TRA-8. Moreover, cytotoxicity of the TRA-8 full-length or Fab' immunoliposome against normal cells, such as human primary hepatocytes, was lower than that for TRAIL. Enhanced activity was also observed for immunoliposomes conjugated with other apoptosis-inducing antibodies against other receptors of the TNFR superfamily, such as death receptor 4 (DR4) and Fas. Thus, immunoliposomes are promising as a new modality that could exhibit significant activity at a low dose, for cost-effective application of an antibody fragment and with stable efficacy independent of the intratumoral environment of patients as a TNF superfamily agonistic therapy.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression.

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Shotgun protein analysis by liquid chromatography-tandem mass spectrometry.

Two-dimensional electrophoresis (2DE) is an excellent technology for the analysis of complex protein mixtures, but it has drawbacks, such as hardly detecting very hydrophobic proteins. Shotgun protein analysis is one of the major technologies used to compensate for the weaknesses of 2DE. In this approach, total proteins are digested as a mixture and the digested peptides are separated by one-dimensional or multidimensional chromatography and introduced into a tandem mass spectrometer. Since the shotgun approach is the primary strategy in proteomics besides 2DE, a great number of related methodologies have been developed. In this chapter, we would like to introduce the simplest protocol, in which proteins are digested in solution and the digested peptides are analyzed by one-dimensional reversed-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), as a starting point for shotgun protein analysis.

Efficient antibody production upon suppression of O mannosylation in the yeast Ogataea minuta.

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones.

A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta.

Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics.

Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.

Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 A resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 A based on a comparison of the crystal structure with the predicted structure. The CDR-H3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the "H3-rules." The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.

Sorafenib and its derivative SC-49 sensitize hepatocellular carcinoma cells to CS-1008, a humanized anti-TNFRSF10B (DR5) antibody.

BACKGROUND AND PURPOSE: Previously, we have shown that sorafenib sensitizes hepatocellular carcinoma (HCC) to apoptosis induced by TNF-related apoptosis-inducing ligand (TNFSF10; TRAIL). Here, we report that sorafenib and SC-49 sensitize HCC cells to CS-1008, a novel anti-human death receptor 5 (TNFRSF10B) antibody. EXPERIMENTAL APPROACH: HCC cell lines (PLC5, Huh-7, and Hep3B) were treated with CS-1008 and/or sorafenib and analysed in terms of apoptosis and signal transductions. KEY RESULTS: SC-49 is a sorafenib derivative, which is devoid of kinase inhibitory activity. Both sorafenib and SC-49 down-regulated the phosphorylation of STAT3 at Tyr(705) and subsequently reduced the levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in CS-1008-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to CS-1008 in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effects of sorafenib and SC-49 on CS-1008-induced apoptosis, indicating that inhibition of STAT3 mediates the enhancing effects of these compounds when combined with CS-1008. Importantly, inhibition of SHP-1 by adding a specific SHP-1 inhibitor reduced the effects of SC-49 and CS-1008 on p-STAT3 and apoptosis, whereas co-treatment of CS-1008 with SC-49 increased the activity of SHP-1. These data indicate that the combined effects of CS-1008 and SC-49 on HCC are mediated by SHP-1. Moreover, the combination of CS-1008 and SC-49 inhibited HCC xenograft tumour growth in vivo. CONCLUSIONS AND IMPLICATIONS: Sorafenib and its derivative SC-49 sensitize HCC cells to the antitumour effects of CS-1008 through SHP-1-dependent inactivation of STAT3.

Dovitinib sensitizes hepatocellular carcinoma cells to TRAIL and tigatuzumab, a novel anti-DR5 antibody, through SHP-1-dependent inhibition of STAT3.

Hepatocellular carcinoma (HCC) often displays resistance to recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Dovitinib, a multiple tyrosine kinase inhibitor, and tigatuzumab, a novel humanized anti-human death receptor 5 (DR5) agonistic antibody, are both under clinical investigations in HCC. Here, we report that dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through inhibition of signal transducers and activators of transcription 3 (STAT3). Our data indicate that HCC cells showed significant resistance to TRAIL- and tigatuzumab-induced apoptosis. The combination of dovitinib and tigatuzumab restored the sensitivity of HCC cells to TRAIL- and tigatuzumab-induced apoptosis. Dovitinib down-regulated phospho-STAT3 (Tyr705) (p-STAT3) and subsequently reduced the protein levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA-interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effect of dovitinib on TRAIL-induced apoptosis. Importantly, silencing SHP-1 by RNA-interference reduced the effects of dovitinib and TRAIL on p-STAT3 and apoptosis, whereas co-treatment of TRAIL and dovitinib increased the activity of SHP-1. Moreover, in vivo the combination of tigatuzumab and dovitinib inhibited Huh-7 xenograft tumor growth. In conclusion, dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through a novel machinery: SHP-1 dependent STAT3 inhibition.

Protective effects of HFE7A, mouse anti-human/mouse Fas monoclonal antibody against acute and lethal hepatic injury induced by Jo2.

HFE7A is a mouse anti-human/mouse Fas monoclonal antibody which, protects mice from fulminant hepatitis induced by Jo2. Herein, we report on the mechanism of the protective effect of HFE7A against Jo2-induced acute and lethal hepatic injury. HFE7A reduced the serum aminotransferase level which was elevated after Jo2 injection. HFE7A also inhibited caspase activation and mitochondrial depolarization in hepatocytes derived from apoptosis induced by Jo2 injection. The protective effect of HFE7A against Jo2-induced apoptosis in mouse hepatocytes was reproducible in vitro. The cell death and caspase activation in isolated mouse hepatocytes were induced by incubating these cells with Jo2 in vitro, and HFE7A inhibited the cell death and caspase activation in mouse hepatocytes in a dose-dependent manner. The affinity of HFE7A to mouse Fas was lower than that of Jo2. The binding of Jo2 to neither recombinant mouse Fas nor mouse hepatocytes was inhibited by an excessive amount of HFE7A. Interestingly, HFE7A bound to hepatocytes isolated from Fas knockout mice. From these results, it is suggested that HFE7A may exert a protective effect against Jo2-induced hepatitis not by competitively inhibiting the binding of Jo2 to Fas on hepatocytes, and that a distinct molecule other than Fas may possibly be involved in the protective effect of HFE7A against Jo2-induced hepatic injury.

A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells.

Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells. The Fas-mediated signaling pathway is classified into two types, type I and type II, in human lymphoid cell lines. We investigated whether a humanized anti-human Fas mAb, R-125224, has cell selectivity in induction of apoptosis. R-125224 induced apoptosis in H9 cells, SKW6.4 cells and activated human lymphocytes when cross-linked with anti-human IgG. On the other hand, R-125224 did not induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. By analysis of death-inducing signaling complex formation, it was demonstrated that R-125224 induced apoptosis selectively in type I cells but not in type II cells. Type I cells also expressed more Fas and had more Fas-clustering activity than type II cells. Moreover, co-localization of these clusters and GM1, which is an sphingoglycolipid associated with lipid rafts, was detected. It was also shown that R-125224 treatment could reduce the number of activated human CD3+Fas+ cells in a SCID mouse model in vivo. Thus, we demonstrated that R-125224 induces apoptosis specifically in type I cells in vitro and in vivo.

Determination of sites citrullinated by peptidylarginine deiminase using 18O stable isotope labeling and mass spectrometry.

Peptidylarginine deiminase (PADI) is an enzyme which catalyzes conversion of arginine residues into citrulline residues in proteins. Citrullination is known to be related to autoimmune diseases including rheumatoid arthritis. Previous work in this laboratory succeeded in identifying citrullinated sites of human fibrinogen by mass spectrometry, but discrimination between citrullination and deamidation of asparagines and glutamine required time-consuming and labor-intensive inspection of tandem mass spectra. In this work a stable isotope is utilized to improve on a previous method for the determination of citrullinated sites by mass spectrometry. Since an oxygen atom is incorporated into the citrulline residue from H(2)O in citrullination by PADI, peptides citrullinated in 50% H(2)(18)O would show a characteristic isotope distribution different from natural abundance, and thus determination of citrullinated sites is expected to be much easier. To verify the utility of this new method, the sites of citrullination of human fibrinogen by human PADI4 were investigated using 50% H(2)(18)O. Compared with the previous method, this new method identified citrullinated sites more easily and effectively, while both the determined citrullinated sites and protein sequence coverage were unaltered.

Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.

Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2',5'-oligoadenylate synthetases (2',5'-OASs), inactivated by 5'-phosphatase and completely degraded by 2'-phosphodiesterase (2'-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2',5'-OASs and RNase L have been molecularly cloned and studied well, the identification of 2'-PDE has remained elusive. Here, we describe the first identification of 2'-PDE, the third key enzyme of the 2-5A system. We found a putative 2'-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2'-PDE by mass spectrometry. Based on the full-length sequence of the human 2'-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2'-PDE expressed in mammalian cells certainly cleaved the 2',5'-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2'-PDE were found, the human 2'-PDE was considered to be a unique enzyme without isoform. Suppression of 2'-PDE by a small interfering RNA and a 2'-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2'-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2'-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis.

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.

Humanization of the mouse anti-Fas antibody HFE7A and crystal structure of the humanized HFE7A Fab fragment.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. We have previously obtained the mouse anti-Fas antibody HFE7A (m-HFE7A), which specifically induces apoptosis in inflammatory cells. In order to apply m-HFE7A for human therapy, we performed antibody humanization of m-HFE7A by grafting the mouse complementarity-determining regions (CDRs) to a human antibody. Five versions of humanized HFE7A (h-HFE7A) demonstrated the same antigen-binding affinity and same competition-binding activity against Fas as the chimeric HFE7A. Furthermore, these h-HFE7As induced the same degree of apoptosis in WR19L12a cells that express human Fas on their surface as chimeric HFE7A does. To further probe the structural basis for antibody humanization, we determined the three-dimensional structure of the h-HFE7A antigen-binding fragment (Fab) by X-ray crystallography and compared it with the crystal structure of the parent m-HFE7A Fab previously determined. The main-chain conformation in each h-HFE7A CDR is almost identical to that in m-HFE7A with root mean square (rms) deviations of 0.14-0.77 A. However, a significant segmental shift was observed in the CDR-L1 loop. Together with the high temperature factors of the CDR-L1 residues, both the loops are flexible, suggesting that the CDR-L1 loop would undergo conformational change upon binding to the antigen. Our results indicate that the humanization of m-HFE7A succeeded in maintaining the main-chain conformation as well as the flexibility of the CDR loop.