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In multiple sclerosis plaques, oligodendroglial connexin (Cx) 47 constituting main gap junction channels with astroglial Cx43 is persistently lost. As mice with Cx47 single knockout exhibit no demyelination, the roles of Cx47 remain undefined. We aimed to clarify the effects of oligodendroglia-specific Cx47 inducible conditional knockout (icKO) on experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55) in PLP/CreERT;Cx47fl/fl mice at 14 d after tamoxifen injection. Cx47 icKO mice demonstrated exacerbation of acute and chronic relapsing EAE with more pronounced demyelination than Cx47 flox (fl)/fl littermates. CD3+ T cells more abundantly infiltrated the spinal cord in Cx47 icKO than in Cx47 fl/fl mice throughout the acute to chronic phases. CXCR3-CCR6+CD4+ and IL17+IFNγ-CD4+ helper T (Th) 17 cells isolated from spinal cord and brain tissues were significantly increased in Cx47 icKO mice compared with Cx47 fl/fl mice, while MOG35-55-specific proliferation and proinflammatory cytokine production of splenocytes were unaltered. Microarray analysis of isolated microglia revealed stronger microglial activation toward proinflammatory and injury-response phenotypes with increased expressions of chemokines that can attract Th17 cells, including Ccl2, Ccl3, Ccl4, Ccl7, and Ccl8, in Cx47 icKO mice compared with Cx47 fl/fl mice. In Cx47 icKO mice, NOS2+ and MHC class II+ microglia were more enriched immunohistochemically, and A1-specific astroglial gene expressions and astroglia immunostained for C3, a representative A1 astrocyte marker, were significantly increased at the acute phase, compared with Cx47 fl/fl mice. These findings suggest that oligodendroglia-specific Cx47 ablation induces severe inflammation upon autoimmune demyelination, underscoring a critical role for Cx47 in regulating neuroinflammation.

Objective Hepatitis C virus (HCV) is associated with B cell lymphoproliferative disorders, including mixed cryoglobulinemia (MC) vasculitis and B cell non-Hodgkin's lymphoma. The expansion of clonal and autoreactive rheumatoid factor–bearing CD21−/low marginal zone (MZ) B cells was demonstrated in patients with HCV-associated MC vasculitis. Fc receptor–like (FCRL) proteins comprise a family of immunoregulatory proteins preferentially expressed on B lineage cells. The goal of this study was to investigate the expression of FCRL proteins 1–5 on B cells from patients with HCV-associated MC vasculitis. Methods Expression of FCRL proteins 1–5 was assessed by flow cytometry on B cells from 15 HCV-infected patients with type II MC (7 of whom had B cell non-Hodgkin's lymphoma), 20 HCV-infected patients without MC, and 20 healthy donors. To evaluate FCRL-5 as an immunotherapy target in HCV-associated MC vasculitis, 2 anti–FCRL-5 recombinant immunotoxins were produced using anti–FCRL-5 monoclonal antibodies and Pseudomonas exotoxin. Results Expression of FCRLs 2, 3, and 5 was markedly increased while expression of FCRL-1 was decreased on clonal CD21−/low MZ B cells, as compared with other B cell subsets, from HCV-infected patients and healthy donors. However, there was no difference in the pattern of FCRL expression between HCV-MC patients with lymphoma and those without lymphoma. The anti–FCRL-5 immunotoxins showed specific cytotoxicity against FCRL-5–expressing clonal CD21−/low MZ B cells isolated from HCV-infected patients as well as FCRL-5–transfected cell lines. No cytotoxicity against T cells or conventional B cells was observed. Conclusion These findings suggest that FCRL-5–targeting therapies could be a specific treatment for HCV-associated MC vasculitis and other FCRL-5–positive autoimmune B cell disorders.

Purpose To prove that chromatin immunoprecipitation assay can be performed with oral rinse samples and to develop a protocol for comprehensive analysis of functional interactions among DNA methylation, histone modification, and gene expression using such samples. Materials and Methods Eleven cancer cell lines and oral rinse samples from 10 patients with oral squamous cell carcinoma and 3 healthy subjects were examined. The expression of CDKN2A , a tumor suppressor gene, was determined by reverse transcription/polymerase chain reaction and immunohistochemistry. Promoter DNA methylation was assessed by methylation-specific polymerase chain reaction. Chromatin modifications were analyzed by a chromatin immunoprecipitation assay using antibodies for dimethylation and acetylation of lysine 9 of histone H3. Results Epigenetic control of CDK2NA was observed in vitro in 11 cancer cell lines. Using the present protocol, comprehensive epigenetic analysis could be successfully performed with oral rinse samples. All patients were comfortable using the prescribed amount (16 mL) of normal saline to rinse their mouths. Nine patients (90%) and 1 healthy subject (33%) showed dimethylation of lysine 9 of histone H3. Moreover, 8 patients (80%) showed hypoacetylation of lysine 9 of histone H3, which was not observed in healthy subjects. Conclusions The present study showed for the first time that chromatin modifications can be analyzed using oral rinse samples by chromatin immunoprecipitation analysis. To evaluate the contribution of histone modifications for carcinogenesis of oral squamous cell carcinoma, studies including a larger number of subjects should be conducted in the future.

BACKGROUND: The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor-related genes with the objective of establishing a noninvasive method for the detection of OSCC. METHODS: Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation-specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene’s methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection. RESULTS: Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E-cadherin (ECAD), transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains 2 (TMEFF2), retinoic acid receptor beta (RARb), and O-6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARb, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT. CONCLUSIONS: The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC. Cancer 2012;118:4298-308. V C 2012 American Cancer Society.

Fc receptor-like 3 (FCRL3) is a cell surface protein homologous to Fc receptors. The FCRL3 gene is present in humans but not in mice. We found that FCRL3 protein is expressed on 40% of human naturally occurring CD4+ regulatory T (nTreg) cells (CD4+CD25+CD127low). Sorted nTreg cells with the surface phenotype FCRL3+ and FCRL3− were both hypoproliferative to TCR stimulation and both suppressive on proliferation of conventional T cells (CD4+CD25−) in vitro. They both expressed forkhead box p3 (Foxp3) protein, the intracellular regulatory T cell marker. However, in contrast to FCRL3− nTreg cells, FCRL3+ nTreg cells were not stimulated to proliferate by the addition of exogenous IL-2. In addition, Foxp3+ cells induced from conventional T cells by TGF-β treatment did not exhibit FCRL3 expression. These results suggest that the FCRL3+ subset of human nTreg cells identified in this study arise in vivo and Foxp3 expression alone is not sufficient to induce FCRL3 expression. FCRL3 may be involved in human-specific mechanisms to control the generation of nTreg cells.

CD22 is an important immunotherapeutic target on B-cell malignancies, particularly hairy cell leukemia (HCL), but its soluble extracellular domain, sCD22, has not yet been reported in the blood. By immunoaffinity and enzyme-linked immunosorbent assay techniques using anti-CD22 monoclonal antibodies, we identified the 100-kDa extracellular domain of CD22 and an 80-kDa processed form in serum of patients with HCL. The median sCD22 level measured by enzyme-linked immunosorbent assay was 18 ng/mL for 93 patients with HCL. sCD22 levels varied from 2.1 to 163 ng/mL and were higher ( P < .001) than 23 normal donors (median, 0.6 ng/mL). More than 95% of normal donors had sCD22 levels less than 1.9 ng/mL. sCD22 levels were proportional to concentrations of circulating HCL cells ( P = .002), and HCL spleen size ( P < .001). sCD22 levels normalized with complete but not partial response to treatment. sCD22 levels up to 300 ng/mL had less than a 2-fold effect on the cytotoxicity of the anti-CD22 recombinant immunotoxin BL22. sCD22 levels may be useful to follow in patients with HCL and may be more specific than sCD25 in patients with CD22+/CD25− disease. Trials are listed on [www.cancer.gov][1] as [NCT00002765][2], [NCT00021983][3], [NCT00074048][4], [NCT00085085][5], [NCT00337311][6], and [NCT00462189][7]. [1]: http://www.cancer.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00002765&atom=%2Fbloodjournal%2F112%2F6%2F2272.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00021983&atom=%2Fbloodjournal%2F112%2F6%2F2272.atom [4]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00074048&atom=%2Fbloodjournal%2F112%2F6%2F2272.atom [5]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00085085&atom=%2Fbloodjournal%2F112%2F6%2F2272.atom [6]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00337311&atom=%2Fbloodjournal%2F112%2F6%2F2272.atom [7]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00462189&atom=%2Fbloodjournal%2F112%2F6%2F2272.atom

Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22–8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.

The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3–4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

NGEP is a prostate-specific gene identified by analysis of expressed sequence tag databases. RNA analysis revealed two spliced forms of NGEP mRNA: a short form encoding a soluble protein (NGEP-S) and a long form encoding a polytopic membrane protein (NGEP-L). Transient expression of myc epitope–tagged NGEP-L showed that it was localized to the plasma membrane. We have now produced a specific antibody to the COOH terminus of NGEP-L and showed that it detects an ∼100-kDa protein in extracts of normal prostate and prostate cancers that contain high levels of NGEP mRNA. The antibody detects a protein that is highly expressed on the apical and the lateral surfaces of normal prostate and prostate cancer cells by immunohistochemistry. The antibody does not detect a protein in the prostate cancer cell line LNCaP, which has very low NGEP mRNA levels. To study NGEP function, two stable LNCaP cell lines were prepared by transfection with NGEP-L and shown to contain similar amounts of NGEP-L protein as human prostate. Confocal immunofluorescence showed that NGEP-L is present on the plasma membrane of the transfected LNCaP cells and is highly concentrated at cell:cell contact regions. Furthermore, as the cell density increased, the cells formed large aggregates. A specific RNA interference that lowered NGEP-L levels prevented formation of cell aggregates. Our results suggest that NGEP-L has a role in promoting cell contact–dependent interactions of LNCaP prostate cancer cells and also that NGEP is a promising immunotherapy target for prostate cancer. [Cancer Res 2007;67(4):1594–601]

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade including, ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility and invasion of PDAC cells - all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13-HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13-HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Purpose: To establish monoclonal antibodies (mAb) against megakaryocyte potentiation factor (MPF) and detect MPF in the blood of patients with mesothelioma. Experimental Design: Mice were immunized with a purified recombinant human MPF-rabbit-Fc fusion protein and with MPF. Several hybridomas producing mAbs to MPF were established. A double-determinant (sandwich) ELISA was constructed using mAbs to two different epitopes and used to determine if MPF is present in the serum of patients with mesothelioma. Results: We established seven anti-MPF mAbs whose topographical epitopes were classified into three nonoverlapping groups. All the mAbs reacted with recombinant MPF protein by ELISA. One of the mAbs detected MPF and the mesothelin precursor protein containing MPF in cell lysates on Western blotting. A sandwich ELISA using mAbs to two different epitopes was constructed and used to measure the presence of MPF in the media of various mesothelin-expressing cancer cell lines and in human serum. The ELISA showed that MPF levels were elevated in 91% (51 of 56) of patients with mesothelioma compared with healthy controls. Furthermore, serum MPF fell to normal levels in two patients after surgery for their peritoneal mesothelioma. Conclusions: Using new mAbs to MPF, we showed that MPF is secreted by several mesothelioma cell lines and is frequently elevated in the blood of patients with mesothelioma. Measurement of MPF may be useful in following the response of mesothelioma to treatment.

Purpose: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors. Experimental Design: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein. Results: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells. Conclusion: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.

Because CD30 is highly expressed on Hodgkin's lymphoma and anaplastic large cell lymphoma, it is a promising target for immunotherapy. Soluble CD30, the extracellular domain of CD30 that is shed from the cells, can reduce the effects of CD30-targeting agents by competitive binding. In this study, we identified two epitopes on membrane-associated CD30 that are missing on soluble CD30 probably because of a conformational change upon shedding. These epitopes are potentially superior targets for immunotherapy because targeting them should be free from the competitive effects of soluble CD30. We studied 27 anti-native CD30 mAbs that were assigned to 8 different topographical epitopes. Soluble CD30 was prepared from culture supernatants of L540 cells or Karpas 299 cells. In an ELISA, the mAbs to two epitopes, Ep2 (amino acids 107–153) and Ep7 (amino acids 282–338), showed less than a 2% average cross-reactivity to soluble CD30 compared with a CD30-Fc fusion protein. In addition, these mAbs bound to CD30 on cells in the presence of an excess of soluble CD30. These epitopes (Ep2 and Ep7) are, therefore, more efficiently presented on cell-associated CD30 than on soluble CD30 (membrane-specific epitopes). Also, soluble CD30 in the sera of mice bearing L540 tumors did not form immune complexes with the membrane-specific mAbs analyzed by size-exclusion chromatography. In contrast, mAbs to the other epitopes reacted with both soluble CD30 and membrane CD30. Our results suggest that it may be possible to find membrane-specific epitopes on other immunotherapy target molecules.

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).

The aim of this study was to examine tumor necrosis factor-α (TNF-α) expression and Kupffer cell activation in hepatotoxicity caused by microcystin-LR (MCLR). Mice received a single intraperitoneal injection of 60.0 μg/kg MCLR and were killed at several time points within 24 hours. MCLR caused hemorrhage within 7 hours, followed by hepatocellular necrosis and apoptosis. A real-time quantitative reverse-transcription polymerase chain reaction demonstrated that the level of TNF-α mRNA was 2.3-fold higher than in the controls at 17 hours. The number of TNF-α-immunopositive nonparenchymal cells was 2.6- and 7.7-fold greater than that in the controls at 7 and 17 hours, respectively, and they frequently infiltrated into necrotic areas, probably in association with neutrophil recruitment. Some apoptotic hepatocytes were immunopositive for cleaved caspase-3. To inactivate Kupffer cells, mice were pretreated with a single intravenous injection of gadolinium chloride (GdCl3, 2 mg/mouse). That brought about the apoptosis of sinusoidal cells, which indicated Kupffer cell depletion. GdCl3 pretreatment attenuated MCLR-induced hepatocellular apoptosis by 68%, and likely decreased hepatocellular necrosis. These results suggest that Kupffer cell activation, represented by enhanced TNF-α expression, is involved in the progression of MCLR-induced hepatotoxicity in mice.

Chemical modification of proteins with polyethylene glycol (PEGylation) can increase plasma half-lives, stability, and therapeutic potency. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of anti-Tac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the IL-2 receptor α subunit fused to a 38-kDa fragment ofPseudomonasexotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then site-specifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activitiesin vitrobut superior stability at 37°C in mouse serum, a 5- to 8-fold increase in plasma half-lives in mice, and a 3- to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans.

Phosphatidylinositol (PI) 3,4-P(2) is a phosphoinositide that has been shown to be important for signal transduction in growth factor stimulation. We have produced monoclonal antibodies specific for PI 3,4-P(2), which were able to detect PI 3,4-P(2) generated in 293T cells treated with H(2)O(2), or in MKN45/BD110 cells expressing activated PI 3-kinase in immunostaining. Prolonged treatment with 0.05% Tween 20 resulted in detection of staining not only at the plasma membrane, but also at the nuclear surface, indicating that 3'-phosphorylated phosphoinositides can be generated and function in the nucleus.

To develop an ultrasensitive immunoassay for microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, we produced monoclonal antibodies (MAbs) which specifically recognize the immune complex (IC) formed by an anti-MC MAb (MC MAb) and MCs. The use of the anti-IC MAb (IC MAb) as the secondary antibody made it possible to develop a sandwich type immunoassay, which is theoretically superior to the widely used competitive immunoassay in sensitivity as well as accuracy. A MC MAb mixed with microcystin-LR (MCLR) to form the IC was immunized to mice. Three IC MAbs were obtained, all of which specifically reacted with the IC, but almost never reacted to MC MAb or MCLR in enzyme-linked immunosorbent assays (ELISAs). Binding kinetics study of one of the IC MAbs, 3F7, by a BIAcore biosensor technique revealed that 3F7 IC MAb could associate with free MC MAb as well as the IC, but the binding to free MC MAb was much more easily dissociated than that to the IC, thus resulting in about 300-fold higher affinity of 3F7 for the IC than for MC MAb alone (1.8 x 10(9) M(-1) and 4.6 x 10(6) M(-1) for the IC and MC MAb, respectively). Finally, 3F7 IC MAb was shown to react with the IC formed by the addition of MCLR to MC MAb-coated plates in a dose-dependent manner. Therefore, a new type sandwich immunoassay, anti-immune complex ELISA (IC ELISA) for MCs, was indeed established. The detection limit of the IC ELISA was 2 pg of MCLR ml(-1) (50 fg per assay), making it the most sensitive of all the methods for detecting MCs reported to date.

PIP3BP is a phosphatidylinositol 3,4,5-trisphosphate-binding protein (PIP3BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP3BP antibody and determination of localization of PIP3BP fused to the green fluorescent protein (GFP-PIP3BP) revealed that PIP3BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP3BP. Generation of PIP3 in the nucleus was detected in H2O2-treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus and enhanced activity of PI 3-kinase in the nucleus fraction were observed after H2O2 treatment of 293T cells, suggesting that PI 3-kinase can be activated in the nucleus as well as in the membrane after appropriate stimulation of the cells. Co-expression of the constitutively active PI 3-kinase with PIP3BP resulted in exportation of the protein from the nucleus to the cytoplasm, suggesting that PIP3BP can function as a PIP3-binding protein in the intact cells. These results imply that there may be an unknown function of PI 3-kinase in the nucleus.

Phosphatidylinositol (PI) 3-kinase is known as one of the key molecules involved in the various biological events such as vesicle trafficking, cytoskeletal rearrangements and cell survival. To clarify the molecular basis underlying these events, we have tried to identify the proteins that can interact with phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), the lipid product of PI3-kinase. Using a new PIP 3 analogue, PIP 3 -APB, we synthesized an affinity column for PIP 3 binding proteins. This enabled us to purify and identify several PIP 3 binding proteins such as Tec tyrosine kinase, Gap1 m , and Akt, as the candidates for the downstream molecules of PI3-kinase. All of these proteins contain PH domains, possible binding sites for phospholipids. Studies with various deletion mutants of Tec or Gap1 m revealed that their PH domains are indeed the binding sites for PIP 3 . These results demonstrate that this PIP 3 analogue binds various PIP 3 binding proteins with high specificity and may be useful to elucidate the downstream mechanisms of PI3-kinases-mediated signaling pathways.

Microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, are suspected as tumor-promoter contaminants of environmental water. We have previously developed an enzyme-linked immunosorbent assay (ELISA) for MCs based on an anti-MC MAb (MAb-mc). We describe here the production of anti-idiotype MAbs (MAb-ids) which react with MAb-mc and their use in a new ELISA for MCs. For the production of MAb-id, hybridoma cells were generated from mice immunized with MAb-mc. Two MAbs were selected for their ability to inhibit the binding of MAb-mc to microcystin-LR (MCLR)-bovine serum albumin conjugate in ELISA. The one with the higher inhibitory activity, designated Id7 (IgG1, kappa), was further characterized. ELISA and immunoprecipitation analysis revealed that Id7 specifically bound to MAb-mc but not to control IgG1, and the binding was inhibited by free MCLR. Therefore, Id7 is a MAb-id to MAb-mc and potentially possesses the structural image of MCLR. To establish MAb-id based ELISA, Id7 was tested for use in three types of competitive ELISA for MCs. The best format enabled reliable measurements of MCLR in the range of 100-1000 pg/ml with a coefficient of variation of less than 3%. In addition, microcystin-RR and microcystin-YR, principal MCs found in environmental water, were cross-reacted well (67-111% of MCLR) in the ELISA although 6(Z)-MCLR, a minor component, was less reactive (7% of MCLR). A comparative study of the MAb-id based ELISA with previously established MAb-mc based ELISA revealed good correlation (n = 14, r = 0.97) between the two methods for measurements of MCs content in freshwater samples. Thus, developed MAb-id based ELISA is an useful alternative for environmental monitoring of MCs.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Fc receptor–like (FCRL) 5 regulates B cell Ag receptor signaling and has been reported to bind aggregated IgG. Using surface plasmon resonance, we analyzed the interaction of native IgG samples with FCRL5, revealing a complex binding mechanism, where isotype is just one factor. FCRL5 bound IgG1 and IgG4 with ∼1 μM KD, whereas the interaction with IgG3 was a magnitude weaker. However, IgG2 samples displayed a wide range of affinities, indicating that additional factors affect binding. We used a panel of 19 anti-FCRL5 mAbs with defined reactivity to identify domains involved in ligand binding. Six mAbs blocked IgG binding, indicating critical roles of FCRL5 domains 1 and 3, as well as epitopes at the domain 1/2 and domain 2/3 boundaries. We found that only glycosylated IgG containing both Fab arms and the Fc region bound with high affinity. Furthermore, the presence of sialic acid in the IgG carbohydrate altered FCRL5 binding. The interaction of IgG and FCRL5 consisted of two kinetic components, suggesting a complex binding mechanism. We established that the IgG-Fc and IgG-F(ab′)2 fragments bind FCRL5 independently but with low affinity, revealing the mechanism behind the two-step binding of whole IgG. This complex binding mechanism is distinct from that of Fc receptors, which bind through the Fc. We propose that FCRL5 is a new type of receptor that recognizes intact IgG, possibly enabling B cells to sense Ig quality. Recognition of undamaged IgG molecules by FCRL5 could allow B cells to engage recently produced Abs.

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, KD = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Mucin 13 (MUC13) is a high-molecular-weight transmembrane glycoprotein that is frequently and aberrantly expressed in a variety of epithelial carcinomas, including gastric, colorectal, and ovarian cancers. On the basis of the high expression of MUC13 in cancer cells as well as recent laboratory findings suggesting a malignant phenotype of MUC13-transfected cell lines, the oncogenic potential of MUC13 has emerged. The various functional domains of MUC13 may confer oncogenic potential to MUC13. For example, the bulky extracellular domain with extensive modification with glycan chains may prevent cell–cell and cell–extracellular matrix binding whereas the cytoplasmic tail containing serine and tyrosine residues for potential phosphorylation may participate in cell signaling. MUC13 exhibits the characteristics suitable as an early marker for cancer screening and presents a promising target for antibody-guided targeted therapy. Mol Cancer Res; 9(5); 531–7. ©2011 AACR.

The SH2 region of signal-transducing proteins mediates binding to phosphotyrosine-containing proteins. We analyzed the structure-function relationship of the SH2 region of the human CRK protein by using a series of monoclonal antibodies (mAbs). Seventeen mAbs against the CRK SH2 region were classified into 5 groups according to the reactivity with mutant CRK proteins expressed in COS7 cells and in Escherichia coli and by epitope scanning with synthetic nonapeptides. Two groups of mAbs (groups A and B) were reactive only with intact SH2. Mutation(s) in either the amino-terminal B box or the carboxyl-terminal C box, which are the two subdomains of SH2, abolished the reactivity of the CRK mutants to the mAbs of groups A and B. Group A mAbs competed the binding of the CRK SH2 region to the phosphotyrosine-containing proteins. Moreover, the spectrum of the CRK mutants which were recognized by group A mAbs coincided with that of the CRK mutants which could bind phosphotyrosine-containing proteins, suggesting that group A mAbs were directed against the site of binding to phosphotyrosine-containing proteins. Group C mAbs, directed against the region between the B and C boxes, were reactive with both wild-type and mutant CRK proteins and did not affect the capacity of SH2 to bind phosphotyrosine-containing proteins. Contrary to group A and B mAbs, mAbs belonging to groups D and E, which were mapped onto the C box, did not bind well to the native CRK proteins, but bound to CRK mutants with mutation(s) in either the B or the C box. These results suggest that the B and C boxes, which are separated by a hinge region, coordinately form the functional SH2 domain that binds to the phosphotyrosine-containing proteins and to the group A mAbs.

Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.

New gene expressed in prostate (NGEP) is a prostate-specific polytopic membrane protein found at high concentrations at cell:cell contact regions. To determine if NGEP is a useful target for antibody-based therapy of prostate cancer, we performed an immunohistochemical analysis of 126 human prostate carcinoma samples using polyclonal anti-NGEP sera and found that 91% of the cancers express NGEP protein. To elucidate the topology of NGEP and guide the development of monoclonal antibodies (mAb) reacting with the extracellular regions of NGEP, a hemagglutinin epitope tag was inserted at several positions within the NGEP sequence. The tagged proteins were expressed in 293T cells and locations of the tags were determined by immunofluorescence in intact or permeabilized cells. The results indicate that NGEP contains eight transmembrane domains with both the NH2 and COOH termini of NGEP located inside the cell. We produced mAb to three regions that are predicted to be intracellular based on the epitope tag data (amino acids 1-352, 441-501, and 868-933), and as predicted, the mAb only detected the protein in permeabilized cells. NGEP is a glycoprotein with predicted glycosylation sites at N809 and N824. When these residues were converted to glutamine, glycosylation was abolished, confirming that the residues are extracellular. Our findings on the expression and the orientation of the NGEP protein serve as an important framework for the development of mAb targeting the extracellular regions of NGEP that could be used for prostate cancer immunotherapy. [Cancer Res 2008;68(15):6306–12]

FCRL1 (Fc receptor–like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC50s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL.

In vitro antibody affinity maturation has generally been achieved by display of mouse or human antibodies on the surface of microorganisms (phage, bacteria, and yeast). However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. An ideal system would select and improve antibodies in a mammalian cell environment where they are naturally made. Here we show that human embryonic kidney 293T cells that are widely used for transient protein expression can be used for cell surface display of single-chain Fv antibodies for affinity maturation. In a proof-of-concept experiment, cells expressing a rare mutant antibody with higher affinity were enriched 240-fold by a single-pass cell sorting from a large excess of cells expressing WT antibody with a slightly lower affinity. Furthermore, we successfully obtained a highly enriched mutant with increased binding affinity for CD22 after a single selection of a combinatory library randomizing an intrinsic antibody hotspot. Important features are that one display selection cycle requires only 1 week, and transfection of cells in a single 100-mm dish produces 10 7 individual clones so that a repertoire of 10 9 is feasible under current experimental conditions.

We have described previously a cDNA library made from membrane-bound polysomal mRNA prepared from breast and prostate cancer cell lines. The library is highly enriched for cDNAs encoding membrane proteins, secreted proteins, and cytokeratins. To characterize this library, 25,277 cDNA clones were sequenced and aligned with various databases; 1,439 clones did not align with known genes. From this set of clones we identified a previously uncharacterized gene encoding a 334-aa protein. Although protein structural motif prediction programs indicate that the gene encodes a membrane protein comprising a signal sequence, a series of leucine-rich repeats, and a single transmembrane domain with a cytoplasmic tail, confocal microscopy of MCF7 breast cancer cells demonstrates that the protein is not directly associated with the plasma membrane or intracellular membranes but instead colocalizes with intermediate filaments and cytokeratins within the cell. Immunofluorescence studies also show that protein expression is increased greatly in mitotic MCF7 cells, and immunohistochemistry demonstrates its expression in human breast cancer cells. Analysis of mRNA levels in 25 different normal tissues by RT-PCR shows that this gene is expressed highly in normal prostate and salivary gland, very weakly in colon, pancreas, and intestine, and not at all in other tissues. RT-PCR studies on human cancer samples show that the RNA is expressed highly in many cancer cell lines and cancer specimens, including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3 colon cancers, and 3 of 3 pancreatic cancers. We name the protein CAPC, cytokeratin-associated protein in cancer.

Background The immunoglobulin superfamily receptor translocation-associated 2 (IRTA2) gene encodes both membrane and secreted forms of the B-cell differentiation antigen (also identified as FcRH5). The membrane form is highly expressed on the surface of hairy cell leukemia (HCL) cells from patients. This study aimed to develop immunoassays for soluble IRTA2/FcRH5 proteins in human serum. Methods Two sandwich ELISAs for soluble IRTA2/FcRH5 were designed using two pairs of monoclonal antibodies specific to four different epitopes on IRTA2/FcRH5. Results In both ELISAs, the lower limit of quantitation for soluble IRTA2/FcRH5 in human serum was 30 ng/mL. The analytical recovery of 0.3-2.1 ng/mL of IRTA2/FcRH5-Fc used as the standard, from a 100-fold dilution of IRTA2/FcRH5-free sera, was 94-106% for ELISA #1 and 89-97% for ELISA #2. Between-assay coefficients of variance were 7.7-17.6% for ELISA #1 and 7.7-32.7% for ELISA #2. Both ELISAs detected soluble IRTA2/FcRH5 protein in sera from normal donors (median 169 ng/mL in ELISA #1 and 146 ng/mL in ELISA #2, n=123) without correlations to gender or age. A marked increase in soluble IRTA2/FcRH5 levels was observed in samples from patients with HCL (medians 719 ng/mL in ELISA #1 and 754 ng/mL in ELISA #2, n=44). Conclusions These ELISAs may be useful for monitoring soluble IRTA2/FcRH5 in HCL and other B-cell malignancies.

ThePATEgene is expressed in prostate and testis. To determine if PATE is expressed in other accessory tissues of the male genital tract, RT-PCR of the epididymis and seminal vesicle was performed. PATE mRNA was highly expressed in the epididymis and seminal vesicle.In situhybridization of the testis showed PATE mRNA is strongly expressed in the spermatogonia. ThePATEgene encodes a 14-kDa protein with a predicted signal sequence and a cleavage site between residues G21 and S22. To determine if PATE is a secreted protein, 293T cells were transfected with a pcDNA-PATE-myc-His plasmid and protein immunoprecipitated with anti-myc monoclonal antibody. Western blot analysis showed the presence of PATE-myc-His protein was in the medium and the cell lysate. Confocal microscopy demonstrated that PATE-myc-His protein is found in the endoplasmic reticulum. The polyclonal antibody SOL-1 was generated by immunization of rabbits with recombinant PATE protein expressed and purified fromEscherichia coli.Western blots were performed on extracts of prostate, testis, seminal vesicle and ejaculated spermatozoa, but PATE protein was only detected in the spermatozoa. Immunostaining of sperm smears revealed that PATE is located in a band-like pattern in the sperm head. Our data indicate that PATE is made by various sexual accessory tissues and secreted into the semen where it becomes associated with sperm, suggesting that PATE is a novel sperm-associated protein with a possible role in mammalian sperm maturation.

Purpose: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue–resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22. Experimental Design: Arg490, which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured. Results: HA22 (R490A) is ∼2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD50s (∼1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity. Conclusion: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.

We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.

Topography of epitopes of monoclonal antibodies (MAbs), identified as the mutual competition of the MAbs, can be valuable indicators for the biological functions of MAbs. However, the determination of topographical epitopes is not performed before the functional screening of MAbs, because the requirement for purifying and labeling of MAbs makes the mapping experiment difficult, particularly in the early stage of MAb production. Here we describe a new label-free competitive enzyme-linked immunosorbent assay (LFC-ELISA) for the rapid grouping of MAbs based on the topography of their epitopes. In the LFC-ELISA, the immune complex formed by a competitor, MAb#2, and an antigen is challenged by an indicator, MAb#1 that had been captured on the ELISA plate through a secondary antibody. The MAb#2-antigen immune complex is trapped by MAb#1 only if MAb#1 reacts with an epitope different from that of MAb#2. The immune complex (MAb#2-antigen-MAb#1) is detected with an enzyme-labeled reagent specific to a tag on the antigen. Our experiments using different anti-CD30 MAbs and a CD30-Fc fusion protein as the antigen revealed that the LFC-ELISA performed well with MAbs of different isotypes (IgG1, IgG2a, and IgG2b), and in a practical range of MAb concentrations (0.3-10 microg/ml) and affinities (0.9-13 nM of Kd). We obtained pairwise competition data from all 26 anti-CD30 MAbs. We then utilized a cluster analysis and a bootstrap method to analyze the competition data for grouping of the MAbs. This objective and automated analysis identified eight distinct topographical epitopes on CD30. The reactivity of the anti-CD30 MAbs in immunoblot, and their inhibiting activity on CD30-CD30-ligand binding correlated with the topographical epitopes. The results show that the LFC-ELISA combined with cluster analysis is a useful new method for grouping MAbs based on their topographical epitopes and can be used in the early stage of MAb production. One useful application is to identify MAbs reacting with different epitopes from a large number of MAbs so that the most appropriate MAbs can be selected for therapeutic use.

Ret is a tyrosine kinase receptor involved in several human diseases germ-line mutations are responsible for multiple endocrine neoplasia type 2 syndromes while somatic mutations of Ret are found in sporadic medullary thyroid carcinomas. In the present work, we describe the generation and characterization of a panel of novel monoclonal antibodies to Ret obtained by immunizing mice with a Ret-FC fusion protein. Fifty-five independent monoclonal antibodies recognize Ret-FC by enzyme linked immunosorbent assay but not a non-related FC fusion protein. Twenty antibodies further characterized recognize Ret expressing cells by flow cytometry. Finally, immunoprecipitation analysis showed that these antibodies recognize Ret mature glycosylated and immature forms. Thus, these monoclonal antibodies could be used as diagnostic tools to detect Ret expression, as well as therapeutic tools to downmodulate Ret or to deliver cytotoxic drugs to malignancies that overexpress Ret as neuroblastomas, medullary and papillary thyroid carcinomas, seminomas, and leukemia.

We have searched for phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP3) binding proteins in Dictyostelium discoideum using beads bearing a PIP3 analogue, PIP3-APB. One of the binding proteins with a molecular mass of 55 kDa was purified and its amino acid sequence was partially analyzed. Database searches showed that the analyzed sequence was identical to that of protein kinase B (PKB) of D. discoideum. The specific activity of D. discoideum PKB, when expressed together with constitutively active PI-3 kinase in mammalian cells, was elevated by about three-fold, suggesting that PKB could also act downstream of PI-3 kinase in Dictyostelium cells.

Phosphatidylinositol (PI)-3 kinase is an enzyme that phosphorylates the D-3 position of PI and its derivatives. It is activated immediately after growth factor or differentiation factor stimulation, suggesting that PI-3 kinase is involved in signal transduction of the stimulation. PI-3 kinase appears to play various important roles including signaling to the nucleus, vesicle transport, and rearrangement of the cytoskeleton since many cell responses which require these events are affected by inhibition or activation of PI-3 kinase. To understand how PI-3 kinase can act in such multiple ways, it is important to identify the factors downstream of PI-3 kinase. In this review, we discuss the factors downstream of PI-3 kinase and the methods used to identify them. Recent studies revealed that some proteins involved in vesicle transport or in rearrangement of the cytoskeleton are regulated by the phospholipids generated by PI-3 kinase, implying the mechanism by which PI-3 kinase regulates these cell responses.

We have explored the levels of phosphatidylinositol-3 kinase protein during culture of rat 3Y1 cells. Confluent cell cultures exhibited a higher level of phosphatidylinositol-3 kinase compared with that of growing cells. After replating of the cells on fresh dishes, the level of phosphatidylinositol-3 kinase returned to that of growing cells within 24h. This density-dependent regulation of the phosphatidylinositol-3 kinase level was not lined to cell growth, because growth arrest by serum starvation did not cause elevation of the phosphatidylinositol-3 kinase level. Northern blotting analysis revealed that this regulation was based on the transcriptional level. After cell growth was arrested by contact inhibition, elevation of the level of phosphatidylinositol-3,4,5-trisphosphate was detected suggesting that phosphatidylinositol-3 kinase was activated in these cells. These effects were not seen in src-transformed 3Y1 cells, suggesting that this regulation was lost in transformed cells.

The amyloid precursor protein (APP) is abnormally cleaved during the progression of Alzheimer's disease, resulting in production of the toxic beta-amyloid peptide, which forms neuritic plaques in the brain. To develop a pharmacological approach for treatment of Alzheimer's disease, natural compounds which may inhibit APP synthesis and/or beta-amyloid production are required. Staurosporine, a toxin isolated from Streptomyces staurospores bacteria, is widely used as a protein kinase C inhibitor in signal transduction research. Using rat pheochromocytoma PC12 sympathetic neurons, which express APP, we characterised staurosporine effect on APP level by western blotting, using an anti-APP monoclonal antibody. PC12 APP levels were increased or decreased upon exposure to either 50-200 nM or 10-20 nM phorbol 12-myristate 13-acetate (PMA, a protein kinase C activator), respectively. An apparent relationship was found between the change in APP level and a differential down regulation process of different PKC isoforms. The PMA-induced increase in intracellular APP level was dose-dependently inhibited by staurosporine (natural alkaloid) or GF 109203X (synthetic analogue), protein kinase C (PKC) inhibitors. This inhibition was mainly observed upon treatment of the cells before the exposure to PMA. These results suggest PKC regulation of APP levels in PC12 cells, and provide staurosporine as a leader compound for the development of drugs to control the expression of APP in Alzheimer's research.

Six monoclonal antibodies (MAbs) to microcystin-LR (MCLR), a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa, were produced. They showed the protective effects on hepatotoxicity of MCLR in vitro and in vivo, and on the inhibition of protein phosphatase by MCLR. Competitive enzyme-linked immunosorbent assays with various microcystins revealed that the six MAbs recognized a part of the molecule, in particular, a tertial structure around Adda, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid. The specificity of these MAbs varied slightly. In primary rat hepatocyte cultures, all MAbs showed protective effects against the MCLR-induced cell damages, assessed by morphological changes, lactate dehydrogenase release into the medium, and a calorimetric assay to measure the cell viability using a tetrazolium dye. The M8H5 MAb showing the highest affinity for MCLR blocked the lethal effects and hepatocellular damage to mice. In addition, M8H5 MAb recovered protein phosphatase 2A inhibition by MCLR in a dose-dependent manner, while phosphatase inhibition by okadaic acid was not affected. Thus, the MAbs specifically reacted with the microcystins and prevented their biological activities. This is the first report on the protective effects of specific monoclonal antibodies on MCLR-induced toxicity. © 1995 Wiley-Liss, Inc.

An 85 kDa subunit (p85 alpha) of phosphatidylinositol 3-kinase (PI-3K) has one SH3 and two SH2 regions [SH2(N) and SH2(C)], which direct protein-protein interaction. We have established eighteen hybridomas producing monoclonal antibodies against p85 alpha to study the structure-function relationship of this protein. Epitope mapping using a series of deletion mutants expressed in E. coli showed that the monoclonal antibodies bound to at least 5 distinct epitope regions, which were well dispersed on p85 alpha except for its carboxyl-terminus. Monoclonal antibodies against amino-terminal regions and polyclonal antibodies against carboxyl-terminal regions immunoprecipitated p85 alpha expressed in human cells and in E. coli. On the other hand, monoclonal antibodies against the central part of p85 alpha failed to immunoprecipitate p85 alpha efficiently; however, they could immunoprecipitate p85 alpha mutants with deletion of either the amino- or the carboxyl-terminal region. Similar results were obtained by immunocytochemistry using confocal microscopy. These results suggested that steric hindrance prevents binding of monoclonal antibodies to the central part of p85 alpha where SH2(N) is located. The SH2(N) may have a distinct function from SH2(C), which is located at the carboxyl-terminal region and has been shown to mediate the binding of PI-3K to activated growth factor receptors.

Vesicular stomatitis virus (VSV)-mediated cell fusion was recently proposed as an alternative fusion technique to generate monoclonal antibody (MAb)-producing hybridomas. In order to further examine this technique, we made direct comparative experiments among VSV, Sendai virus (SV) and polyethylene glycol (PEG)-mediated cell fusion in generating MAb-producing hybridomas. The distribution of immunoglobulin (Ig) isotypes secreted by the hybridomas obtained, as well as hybridoma yield and specific hybridoma yield, was compared. The results show that VSV-fusion yielded almost the same number of specific hybridomas as SV- and PEG-fusion in spite of its lower fusion frequency. In addition, VSV-fusion preferentially gave Ig-producing, especially IgG-producing, hybridomas. SV-fusion yielded both hybridomas and specific hybridomas with similar frequency to PEG-fusion, but IgM-producers predominated. These results demonstrate that fusion method has a considerable influence on the isotypes of obtained antibodies, and also suggest an advantage of VSV-fusion for production of IgG monoclonal antibodies.