Anti­EGFR monoclonal antibody 134­mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo­ or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti­EGFR monoclonal antibody (mAb), EMab­134, by immunizing mice with the ectodomain of human EGFR. EMab­134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab­134 was converted from IgG1 to IgG2a (134­mG2a) to facilitate antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab­134 and 134­mG2a against EGFR­expressing CHO­K1 (CHO/EGFR) cells were determined by flow cytometry to be 3.2x10­9 M and 2.1x10­9 M, respectively; these results indicate that 134­mG2a has a higher binding affinity than EMab­134. The 134­mG2a antibody was more sensitive than EMab­134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134­mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC­2, and SAS cells. Moreover, the in vivo administration of 134­mG2a significantly inhibited the development of CHO/EGFR, HSC­2, and SAS mouse xenografts in comparison to the results observed in response to EMab­134. Taken together, the findings of the present study demonstrate that the newly­formulated 134­mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR­expressing oral cancer.

H2Mab-19, an anti-human epidermal growth factor receptor 2 monoclonal antibody exerts antitumor activity in mouse oral cancer xenografts.

Human epidermal growth factor receptor 2 (HER2) is reported to be overexpressed in breast cancers and is associated with poor clinical outcome. Trastuzumab is a humanized anti-HER2 antibody that offers significant survival benefits to patients with HER2-overexpressing breast cancer. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa) was developed. Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of H2Mab-19 were investigated using both breast cancer and oral cancer cell lines. H2Mab-19 demonstrated cytotoxicity in BT-474 (a human breast cancer cell line) and HSC-2 or SAS (human oral cancer cell lines). H2Mab-19 also possessed both ADCC and CDC activity against BT-474, HSC-2, and SAS cell lines. In comparison to control mouse IgG, H2Mab-19 significantly reduced tumor development in BT-474, HSC-2, and SAS xenografts. Collectively, these results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing breast and oral cancers.

Establishment of C20Mab-11, a novel anti-CD20 monoclonal antibody, for the detection of B cells.

CD20 is one of several B-lymphocyte antigens that has been shown to be an effective target for the detection and treatment of B-cell lymphomas. Sensitive and specific monoclonal antibodies (mAbs) are required for every application used for the diagnosis of B-cell lymphoma. Although many anti-CD20 mAbs have been established, the types of applications, those anti-CD20 can be used in, are limited. In this study, we aimed to establish novel anti-CD20 mAbs to be used for broad applications, such as flow cytometry, western blot, and immunohistochemical analyses, using the Cell-Based Immunization and Screening (CBIS) method. One of the established mAbs, C20Mab-11 (IgM, kappa), detected overexpression of CD20 in CHO-K1 or LN229 cell lines, indicating that C20Mab-11 is specific for CD20. In western blot analyses, C20Mab-11 detected not only overexpression of CD20 in CHO-K1 or LN229 cell lines, but also CD20 of BALL-1 and Raji cells with both sensitivity and specificity. Furthermore, C20Mab-11 strongly stained B cells of the lymph follicle and B cell lymphomas in immunohistochemical analyses. These results indicate that C20Mab-11 develped by CBIS method, is useful for the detection of CD20 in lymphoma tissues by flow cytometry, western blot, and immunohistochemical analyses and potentially could be beneficial for the treatment of B cell lymphomas.

A novel anti-EGFR monoclonal antibody (EMab-17) exerts antitumor activity against oral squamous cell carcinomas via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity.

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases; it is a transmembrane receptor involved in cell growth and differentiation. EGFR homodimers or heterodimers in combination with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many types of cancer, including oral squamous cell carcinoma (OSCC). The present study produced novel anti-EGFR monoclonal antibodies (mAbs) possessing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and investigated antitumor activity. Mice were immunized with an EGFR-overexpressed glioblastoma cell line, LN229 (LN229/EGFR), after which ELISA was performed using recombinant EGFR. mAbs were subsequently selected according to their efficacy for LN229/EGFR, as determined via flow cytometry. After determining the subclass of mAbs, the EMab-17 (IgG2a, kappa) clone exhibited ADCC and CDC activities against two OSCC cell lines, HSC-2 and SAS. Furthermore, EMab-17 exerted antitumor activities against mouse xenograft models using HSC-2 and SAS, indicating that EMab-17 may be used in an antibody-based therapy for EGFR-expressing OSCC.

Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM.

Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin.

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.

Characterization of monoclonal antibody LpMab-3 recognizing sialylated glycopeptide of podoplanin.

Podoplanin (PDPN/Aggrus/T1α/gp36/OTS-8), a type I transmembrane sialoglycoprotein, is involved in platelet aggregation, cell invasion, and cancer metastasis. Podoplanin expression in cancer cells or cancer-associated fibroblasts was reported to be involved in poor prognosis of several cancers. Furthermore, podoplanin is expressed in lymphatic endothelial cells or lung type I alveolar cells. Although many anti-podoplanin monoclonal antibodies (MAbs), such as NZ-1 and D2-40, have been established, almost all anti-podoplanin MAbs are produced against a platelet aggregation-inducing (PLAG) domain. In this study, we produced and characterized a novel anti-podoplanin monoclonal antibody, LpMab-3, the epitope of which is a sialylated glycopeptide of podoplanin. We identified the minimum epitope of LpMab-3 as Thr76-Glu81 of human podoplanin, which is different from PLAG domain, using Western blot analysis and flow cytometry. Immunohistochemical analysis showed that LpMab-3 is useful for detecting lung type I alveolar cells and lymphatic endothelial cells. Because LpMab-3 detects only sialylated podoplanin, it could be useful for uncovering the physiological function of sialylated human podoplanin.

A platform of C-type lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin and nonglycosylated rhodocytin.

Podoplanin is a transmembrane O-glycoprotein that binds to C-type lectin-like receptor 2 (CLEC-2). The O-glycan-dependent interaction seems to play crucial roles in various biological processes, such as platelet aggregation. Rhodocytin, a snake venom, also binds to CLEC-2 and aggregates platelets in a glycan-independent manner. To elucidate the structural basis of the glycan-dependent and independent interactions, we performed comparative crystallographic studies of podoplanin and rhodocytin in complex with CLEC-2. Both podoplanin and rhodocytin bind to the noncanonical "side" face of CLEC-2. There is a common interaction mode between consecutive acidic residues on the ligands and the same arginine residues on CLEC-2. Other interactions are ligand-specific. Carboxyl groups from the sialic acid residue on podoplanin and from the C terminus of the rhodocytin α subunit interact differently at this "second" binding site on CLEC-2. The unique and versatile binding modes open a way to understand the functional consequences of CLEC-2-ligand interactions.

A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin.

Podoplanin (PDPN/Aggrus/T1α), a platelet aggregation-inducing mucin-like sialoglycoprotein, is highly expressed in many cancers and normal tissues. A neutralizing monoclonal antibody (mAb; NZ-1) can block the association between podoplanin and C-type lectin-like receptor-2 (CLEC-2) and inhibit podoplanin-induced cancer metastasis, but NZ-1 reacts with podoplanin-expressing normal cells such as lymphatic endothelial cells. In this study, we established a cancer-specific mAb (CasMab) against human podoplanin. Aberrantly glycosylated podoplanin including keratan sulfate or aberrant sialylation, which was expressed in LN229 glioblastoma cells, was used as an immunogen. The newly established LpMab-2 mAb recognized both an aberrant O-glycosylation and a Thr55-Leu64 peptide from human podoplanin. Because LpMab-2 reacted with podoplanin-expressing cancer cells but not with normal cells, as shown by flow cytometry and immunohistochemistry, it is an anti-podoplanin CasMab that is expected to be useful for molecular targeting therapy against podoplanin-expressing cancers.

Multi-specific monoclonal antibody MsMab-2 recognizes IDH1-R132L and IDH2-R172M mutations.

Mutations of isocitrate dehydrogenase 1/2 (IDH1/2) produce oncometabolite R(-)-2-hydroxyglutarate in several tumors. Arginine 132 (R132) of IDH1 and arginine 172 (R172) of IDH2 are functionally important residues. Although MsMab-1 monoclonal antibody (MAb), which is multi-specific for mutated IDH1/2, has been established, MsMab-1 does not react with all IDH1/2 mutations. Herein, we immunized rats with IDH1-R132L peptide, and screened IDH1-R132L-reactive/IDH1-wild-type non-reactive MAbs in enzyme-linked immunosorbent assay. Unexpectedly, the newly established MsMab-2 MAb recognized not only IDH1-R132L but also IDH2-R172M in Western blot analyses, neither of which was detected by MsMab-1. Taken together, the combination of MsMab-1 and MsMab-2 could be useful in diagnosis of mutated IDH1/2-bearing tumors.

Novel monoclonal antibodies GMab-r1 and LMab-1 specifically recognize IDH1-R132G and IDH1-R132L mutations.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate in cytosol. IDH1 mutations, which are specific to a single codon in the conserved and functionally important Arginine 132 (R132), result in the ability of the enzyme to catalyze the reduced NADP-dependent reduction of α-ketoglutarate to onco-metabolite R(-)-2-hydroxyglutarate (2-HG). IDH1 mutations, which are early and frequent genetic alterations that occur in gliomas, cartilaginous tumors, and leukemias. We previously established two monoclonal antibodies (MAbs) that are specific for IDH1 mutations: clone HMab-1 against IDH1-R132H and clone SMab-1 against IDH1-R132S. However, specific MAbs against IDH1-R132G or IDH1-R132L have not been reported. To establish IDH1-R132G-specific or IDH1-R132L-specific MAbs, we immunized rats with each mutation-containing IDH1 peptides, and IDH1-R132G-specific or IDH1-R132L-specific MAbs were screened in ELISA. Established MAb GMab-r1 reacted with the IDH1-R132G peptide, but not with IDH1-wild type (WT) in ELISA. In contrast, LMab-1 reacted with the IDH1-R132L peptide, but not with IDH1-WT. Western blot analysis also showed that GMab-r1 and LMab-1 reacted with the IDH1-R132G and IDH1-R132L recombinant proteins, respectively, but not with IDH1-WT or other IDH1 mutants, indicating that GMab-r1 and LMab-1 are IDH1-mutation-specific. Furthermore, GMab-r1 and LMab-1 specifically stained the IDH1-R132G- and IDH1-R132L-expressing cells in immunocytochemistry, respectively. This is the first report to establish anti-IDH1-R132G-specific or IDH1-R132L-specific MAbs, which could be useful in the diagnosis of mutation-bearing tumors.

A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

A novel monoclonal antibody GMab-m1 specifically recognizes IDH1-R132G mutation.

Isocitrate dehydrogenase 1 (IDH1) mutations occur in gliomas, acute myeloid leukemias, and cartilaginous tumors. While IDH1 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate in cytosol, mutated IDH1 proteins possess the ability to change α-ketoglutarate into onco-metabolite R(-)-2-hydroxyglutarate (2-HG). To data, two monoclonal antibodies (mAbs), which are specific for IDH1 mutations have been established: clone HMab-1 against IDH1-R132H and clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH1-R132G, which are useful for immunohistochemical analysis, have not been reported. To establish IDH1-R132G-specific mAbs, we immunized mice with IDH1-R132G-containing peptides. Established mAb GMab-m1 reacted with the IDH1-R132G peptide, but not with IDH1-wild type (WT) in ELISA. Western-blot analysis also showed that GMab-m1 reacted with the IDH1-R132G recombinant proteins, not with IDH1-WT or other IDH1 mutants, indicating that GMab-m1 is IDH1-R132G-specific. Furthermore, GMab-m1 specifically stained the IDH1-R132G-expressing glioma cells in immunohistochemistry. This is the first report to establish anti-IDH1-R132G-specific mAbs, which is useful in immunohistochemistry of IDH1-R132G-bearing tumors.

Generation of a novel monoclonal antibody WMab-1 specific for IDH2-R172W mutation.

Isocitrate dehydrogenase 2 (IDH2) mutations have been detected in gliomas, cartilaginous tumors, and acute myeloid leukemias. IDH2 mutations are specific to a single codon in the conserved and functionally important Arginine 172 (R172) or Arginine 140 (R140). To date, we have established specific monoclonal antibodies (mAbs) against IDH2-R172K and IDH2-R172M. However, specific mAbs against IDH2-R172W have not been reported. To establish IDH2-R172W-specific mAbs, we immunized rats with IDH2-R172W peptides. Western-blot analysis showed that WMab-1 reacted with the IDH2-R172W recombinant protein, not with IDH2-wild type (WT) or other IDH2 mutants, indicating that WMab-1 is IDH2-R172W-specific. Furthermore, WMab-1 specifically stained the IDH2-R172W-expressing cells in immunocytochemistry, but did not stain IDH2-WT and IDH2-R172M-containing cells. WMab-1 also specifically stained the IDH2-R172W-expressing glioma cells in immunohistochemistry. This is the first report to establish an anti-IDH2-R172W-specific mAb, which could be useful in the diagnosis of IDH2-R172W-bearing tumors.

Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations.

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors, and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in IDH2. We previously established several monoclonal antibodies (mAbs), which are specific for IDH1 mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-specific mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K and IDH2-R172M. After cell fusion, IDH2 mutation-specific mAbs were screened in Enzyme-Linked Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1 are IDH2-mutation-specific. Furthermore, MMab-1 specifically stained the IDH2-R172M-expressing cells in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In immunohistochemical analysis, MMab-1 specifically stained IDH2-R172M-expressing glioma. This is the first report to establish anti-IDH2-mutation-specific mAbs, which could be useful in diagnosis of mutation-bearing tumors.

Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1.

Somatic mutations of isocitrate dehydrogenase (IDH) 1 and IDH2 occur in gliomas, acute myeloid leukemia, and cartilaginous tumors. Somatic mosaic IDH1/2 mutations are also reported in Ollier disease and Maffucci syndrome, which are characterized by multiple central cartilaginous tumors. Although IDH1/2 mutation analysis against osteosarcoma has been performed in several studies, no IDH1/2 mutation has been reported. Herein, we newly report the IDH2-R172S mutation in three of 12 (25%) osteosarcoma patients, which was detected by direct DNA sequencing. No monoclonal antibody (mAb) has been reported against IDH2-R172S mutation. However, we demonstrate that the IDH2-R172S peptide was recognized by our established multi-specific anti-mutated IDH1/2 mAb, MsMab-1, in enzyme-linked immunosorbent assay. Western blot analysis revealed that MsMab-1 reacts with PA tag combined recombinant proteins of IDH2-R172S. Furthermore, MsMab-1 stained IDH2-R172S-expressing osteosarcoma tissues in immunohistochemistry. The MsMab-1 stained nine of 32 (28.1%) osteosarcomas in a tissue microarray. This report is the first describing IDH2 mutations in osteosarcoma, which can be detected by MsMab-1 mAb. Taken together, these results show that MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing osteosarcoma.

Chimeric anti-podoplanin antibody suppresses tumor metastasis through neutralization and antibody-dependent cellular cytotoxicity.

Podoplanin is a platelet aggregation-inducing factor associated with tumor metastasis, malignant progression, and cancer stem cells. We produced a rat-human chimeric anti-podoplanin mAb, NZ-8, from rat anti-podoplanin mAb (NZ-1). Although both NZ-1 and NZ-8 possess high binding affinities and high neutralizing activities of platelet aggregation, the antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of NZ-8 were much higher than NZ-1. Furthermore, both NZ-1 and NZ-8 inhibited the growth of podoplanin-expressing tumors in vivo. Both NZ-1 and NZ-8 also suppressed hematogenous metastasis of podoplanin-expressing tumors. These results suggest that anti-podoplanin mAbs suppressed hematogenous metastasis by both neutralization and antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity activities. Targeting therapy to podoplanin-expressing tumors should be useful as a novel immunotherapy.

Immunohistochemical detection of IDH1 mutation, p53, and internexin as prognostic factors of glial tumors.

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas, are specific to arginine 132 (R132). Recently, we established monoclonal antibodies (mAbs) against IDH1 mutations: anti-IDH1-R132H and anti-IDH1-R132S. However, the importance of immunohistochemistry using the combination of those mAbs has not been elucidated. For this study, 164 cases of glioma were evaluated immunohistochemically for IDH1 mutations (R132H and R132S) using anti-IDH1 mAbs (HMab-1 and SMab-1). IDH1 mutation was detected, respectively, in 9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV, secondary grade IV, grade III, and grade II gliomas. For each grade of glioma, prognostic factors for progression-free survival and overall survival were evaluated using clinical and pathological parameters in addition to IDH1 immunohistochemistry. IDH1 mutation, p53 overexpression, and internexin expression, as evaluated using immunohistochemistry with clinical parameters such as degree of surgical removal and preoperative Karnofsky Performance Status (KPS), might be of greater prognostic significance than histological grading alone in grade III as well as IDH1 mutation in grade IV gliomas.

Podoplanin expression in advanced atherosclerotic lesions of human aortas.

Thrombus formation on disrupted atherosclerotic lesion is a key mechanism of cardiovascular events. Podoplanin (Aggrus), expressed on the surface of several tumor cells, is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), and is involved in tumor cell-induced platelet aggregation and its malignant potency. Podoplanin, which is also expressed in lymphatic endothelial cells, facilitates blood/lymphatic vessel separation. However, podoplanin expression in atherosclerotic lesion has not been investigated. To clarify podoplanin expression in atherosclerotic lesion and to assess its importance for the onset of cardiovascular events, we examined podoplanin expression in abdominal aortas obtained from 31 autopsy cases. Immunohistochemical analysis indicated that podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smooth muscle cells, compared with early lesions composed of smooth muscle cells and small numbers of macrophages. Furthermore, Western-blot and real time-PCR analyses showed that podoplanin expression was significantly enhanced in advanced atherosclerotic lesions, compared with early lesions. These results suggest that podoplanin contributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel molecular target for an anti-thrombus drug.

Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation.

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.