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Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG2a, kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H2Mab-77 (IgG1, kappa) was selected. Finally, immunohistochemical analyses with H2Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H2Mab-77 to detect HER2 in pathological analyses of breast cancers.

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.

The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol to phosphatidic acid. Since both diacylglycerol and phosphatidic acid serve as signaling molecules, DGK is regarded as a hub between diacylglycerol-mediated and phosphatidic acid-mediated signaling. One of the 10 DGK isozymes, DGKα, is shown to be involved in T cell function. Transfection studies using tagged expression vectors revealed that DGKα localizes to the cytoplasm and nucleus and translocates to the plasma membrane in response to T cell receptor stimulation. However, a limited number of studies reported the localization of native protein of DGKα in tissues and cells. In this study, we immunized mice with recombinant DGKα and developed several anti-DGKα monoclonal antibodies (mAbs). One of the established anti-DGKα mAbs is a clone DaMab-2 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, DaMab-2 recognized only DGKα, and did not react with the other isozymes, such as DGKγ, DGKζ, DGKη, and DGKδ. Importantly, DaMab-2 is very useful in immunocytochemical analysis of human cultured cells, indicating that DaMab-2 is advantageous to analyze the localization and function of DGKα.

Podocalyxin (PODXL) is expressed in several cancers, including brain tumors and colorectal cancers. PODXL overexpression is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells, and produced a clone PcMab-47 that could be used for investigating PODXL expression by flow cytometry and immunohistochemical analysis. Herein, we produced a human-mouse chimeric PcMab-47 (chPcMab-47) and investigated its antitumor activity against PODXL-expressing tumors. chPcMab-47 reacted with LN229, LN229/PODXL, and Chinese hamster ovary (CHO)/PODXL cells, but it did not react with CHO-K1 or PODXL-knockout LN229 cell line (PDIS-13). chPcMab-47 exerted antitumor activity against a mouse xenograft model using CHO/PODXL. Furthermore, chPcMab-47 was reactive with colorectal cancer cell lines such as HCT-15, Caco-2, HCT-8, and DLD-1. chPcMab-47 also exhibited antitumor activity against a mouse xenograft model using HCT-15. These results suggest that chPcMab-47 could be useful for antibody therapy against PODXL-expressing cancers.

Giant cell tumors of bone (GCTB) are benign and locally destructive tumors that include osteoclast-type multinuclear giant cells. No available treatment is definitively effective in curing GCTB, especially in surgically unresectable cases. Isocitrate dehydrogenase (IDH) mutations have been reported not only in gliomas and acute myeloid leukemias, but also in cartilaginous tumors and osteosarcomas. However, IDH mutations in GCTB have not been investigated. The IDH mutations are remarkably specific to arginine 132 (R132) in IDH1 and arginine 172 (R172) or arginine 140 (R140) in IDH2; IDH1/2 mutations are known to convert α-ketoglutarate to oncometabolite R(-)-2-hydroxyglutarate. We recently reported that the most frequent IDH mutation in osteosarcomas is IDH2-R172S, which was detected by MsMab-1, a multispecific anti-IDH1/2 mAb. Herein, we newly report the IDH mutations in GCTB, which were stained by MsMab-1 in immunohistochemistry. DNA direct sequencing and subcloning identified IDH mutations of GCTB as IDH2-R172S (16 of 20; 80%). This is the first report to describe IDH mutations in GCTB, and MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing GCTB.

With the aim of obtain the world's biggest glassy alloy, a molten Pd 42.5 Cu 30 Ni 7.5 P 20 alloy with a total weight of 3432 g was water-quenched in a quartz tube. As a result, a cylindrical glassy alloy sample with a diameter of 80 mm and a length of 85 mm was successfully obtained. X-ray diffraction measurements for crashed fragments reveal that the obtained whole sample is vitrified into complete glassy state except for little irregular region of the bubble surface. In addition, differential scanning calorimetry measurements also reveal that the sample has quite homogeneous structure and stable thermal characteristics. In order to estimate a cooling rate for each location of the sample, finite-difference-method calculations using physical properties previously reported were carried out. The calculations exhibited good agreements with previous results and the present experimental results, verifying the validity of the calculations.

With the aim of applying to a soft-magnetic underlayer of the new type of bit-patterned media using nano-patterned glassy alloy thin film, (CoFe)–B–Si–Nb alloy thin films were deposited on a Si substrate by a DC magnetron sputtering method. The deposited thin films with the compositions of Co77Fe5B15Si2Nb1 and Co75Fe5B14Si5Nb1 were confirmed as a fully amorphous structure. The obtained Co77Fe5B15Si2Nb1 amorphous alloy thin film containing lower metalloid content exhibits extremely smooth surface and good soft-magnetic properties such as high saturation flux density of 1.2 T, in-plane low coercivity of 36 A/m, nearly zero-magnetostriction of 4 × 10−7 and high real part of permeability of 870 at 291 MHz, suggesting that the film is appropriate for the soft-magnetic underlayer. Moreover, a prototype bit-patterned media was prepared by utilizing the obtained thin film as a soft-magnetic underlayer. As a result of static tester measurements with a commercial perpendicular magnetic head, accurate information reading and writing on the prototype bit-patterned media were confirmed. It is therefore said that the Co77Fe5B15Si2Nb1 amorphous alloy thin film is a candidate material for the soft-magnetic underlayer of the new type of bit-patterned media.

Podoplanin, a type I transmembrane protein, is expressed in lymphatic endothelial cells. Although we previously developed an anticanine podoplanin monoclonal antibody (mAb), PMab-38, immunohistochemistry (IHC) showed that it did not react with canine lymphatic endothelial cells. Here, we determined whether PMab-38 recognizes canine podoplanin of squamous cell carcinomas (SCCs) and clarified its epitope. In IHC, PMab-38 reacted with 83% of SCCs (15/18 cases). Flow cytometry showed that the epitope of PMab-38 was different from that of the platelet aggregation-stimulating domain of the N-terminus, which was detected by almost all antipodoplanin mAbs such as D2-40 or NZ-1. PMab-38 is expected to be useful for investigating the function of podoplanin in canine tumors.

With the aim of developing a novel preparation process for bit-patterned-media, nano-patterned glassy alloy composite composed of zero-magneto-striction Co–Fe–B amorphous alloy thin film, Pd–Cu–Ni–P glassy alloy and Co/Pd multi-layer with perpendicular magnetization was prepared. Using the commercial imprinting mold, periodic nano hole array with a hole diameter of 90 nm and a pitch of 180 nm was successfully prepared on the surface of Pd-based glassy alloy thin film. By overlaying the Co/Pd multi-layer with perpendicular magnetization and surface polishing, isolated Co/Pd magnetic dots embedded into nano hole array can be obtained. These magnetic dots act as single magnetic domains. In addition, the change in magnetization direction was confirmed under magnetic fields of ±20 kOe. These results suggest that the glassy alloy nano-composite is suitable for BPM of high data density HDD. It is therefore concluded that the preparation method is candidate process for the preparation of BPM of high data density HDD.

In order to clarify detailed surface morphologies and cross sectional nano-structures of novel prototype bit-patterned-medias using nano-imprinted glassy alloy thin films, observations using scanning and transmission electron microscopes were carried out. By employing a focused ion beam milling with an extra low energy, the delicate and fragile structures of samples were successfully maintained and protected during sample preparations. The sequential observations of the preparation process reveal that Co/Pd multilayer is embedded into the imprinted glassy alloy thin film nano hole array with a hole diameter of 30 nm, and the embedded Co/Pd multilayer as acting magnetic recording dots is isolated each other. Using the obtained results, technological and productive feasibility of the prototype BPM as a next generation HDD with a data density above 2 Tbit/inch 2 is also discussed.

With the aim of investigating nano-imprintability of glassy alloys in a film form, Zr(49)Al(11)Ni(8)Cu(32), Pd(39)Cu(29)Ni(13)P(19) and Cu(38)Zr(47)Al(9)Ag(6) glassy alloy thin films were fabricated on Si substrate by a magnetron sputtering method. These films exhibit a very smooth surface, a distinct glass transition phenomenon and a large supercooled liquid region of about 80 K, which are suitable for imprinting materials. Moreover, thermal nano-imprintability of these obtained films is demonstrated by using a dot array mold with a dot diameter of 90 nm. Surface observations revealed that periodic nano-hole arrays with a hole diameter of 90 nm were successfully imprinted on the surface of these films. Among them, Pd-based glassy alloy thin film indicated more precise pattern imprintability, namely, flatter residual surface plane and sharper hole edge. It is said that these glassy alloy thin films, especially Pd-based glassy alloy thin film, are one of the promising materials for fabricating micro-machines and nano-devices by thermal imprinting.

With the aim of investigating fundamental properties and nano-imprintabilities of glassy alloy in the film form, Zr49Al11Ni8Cu32, Pd39Cu29Ni13P19 and Cu38Zr47Al9Ag6 alloy thin films were fabricated on Si substrate by a magnetron sputtering method. These thin films exhibit distinct glass-tradition phenomenon and large supercooled liquid region of about 80 K, confirming as a glassy structure and have very smooth surface and sufficient hardness to maintain imprinted shape, which are suitable for nano-imprint processing. Moreover, thermal nano-imprintabilies of these obtained films are demonstrated by using a dot array mold with a dot diameter of 90 nm and a pitch of 180 nm. Surface observations revealed that periodic nano-hole arrays were successfully imprinted on the surface of these films and precisely corresponded to the periodic dot pattern of the mold. Particularly, Pd-based glassy alloy thin film indicated more precise pattern imprintability, namely, more flat residual surface plane and sharper hole edge. These results suggest that these glassy alloy thin films, especially Pd-based glassy alloy thin film have high potential for application to the nano-imprinting materials.

Present progress in developments of glassy alloy composites for bit-patterned-media and non-equilibrium Cu-based alloys for conductive materials of electrical connectors are reviewed. It is proven that the imprinting of the Pd-based glassy alloy thin film is favorable for the formation of nano-structured devices. Detailed imprinted morphologies formed by different imprinting conditions were examined. In addition, technology of large area imprinting up to 2.5 inches area has been successfully developed and it is now available for production. These technological developments will be utilized for next generation bit-patterned-media with high data density. A newly developed non-equilibrium Cu-Zr-Ag alloy was prepared into sheet form by the combination of casting, cold rolling and annealing. The alloy sheet exhibited high tensile strength of exceeding 1500 MPa and good electrical conductivity of 30% IACS. However, bending ductility should be improved for the actual production of connector. Through the several examinations, remaining issues that should be solved are discussed in the framework of industrialization and commercialization. These obtained results suggest that the glassy alloy composite or non-equilibrium alloy designed by the glass-forming rules have a great potential to develop innovative products in the near future.

Here, the authors fabricated a mold consisting of nanodot arrays with an 18-nm pitch and performed nanoimprinting of metallic glass for developing bitpatterned media (BPM) with an areal recording density of 2 Tbit/in.2. Specifically, they investigated the feasibility of SiO2/Si mold fabrication by metal mask patterning with focused ion beam assisted chemical vapor deposition (FIB-CVD) and reactive ion etching (RIE). SiO2 was etched with a mixed gas of CHF3 and O2, resulting in successful fabrication of convex nanodot arrays with an 18-nm pitch. The authors attempted nanoimprinting of Pd-based metallic glass with the fabricated SiO2 mold and clearly confirmed the replication of the fine nanohole pattern. These results suggest that the proposed FIB-CVD and RIE process is a promising method for fabricating ultrafine nanodot arrays and that metallic glasses are excellent nanoimprintable materials for mass-produced nanodevices such as BPM with ultrahigh recording density.

Here we attempted to fabricate molds (dies) of nanodot arrays with a 25-nm pitch and to nanoimprint metallic glass for developing bit-patterned media with an ultrahigh recording density of 1 Tbit/in.2. The mold-fabricating process consisted of mask patterning by focused ion beam assisted chemical vapor deposition (FIB-CVD) and reactive ion etching (RIE). We investigated the feasibility of a Pt-deposited metal etching mask on SiO2 on Si and diamond like carbon (DLC) on Al2O3 substrates, and achieved isolated convex nanodot arrays with a 25-nm pitch and an aspect ratio of 1.8 by RIE with O2 plasma on a DLC/Al2O3 substrate. Subsequently, we nanoimprinted Pt-based metallic glass by using the fabricated molds and successfully replicated fine concave nanohole arrays. The results suggest that the FIB-CVD/RIE process is a promising technique for fabricating ultrafine nanopatterned molds, and metallic glasses are ideal nanoimprintable materials for mass producing nanodevices such as bit-patterned media.