Analysis of ras gene expression in stomach cancer by anti-ras p21 monoclonal antibodies

Using anti-ras p21 monoclonal antibodies, RASK-3, which reacts with all of Ki-, N-, and Ha-ras p21, we examined by immunohistochemistry the expression of p21 in human gastric cancer (80 cases) and benign gastric lesions (32 cases). Ten percent formalin fixed tissues were studied. Ras p21 was positive in 51 cases (64%) out of 80 cases and partially positive in 12 cases (15%) at the cancerous areas. Ras p21 was partially positive in 7 cases (9%) at the noncancer areas of the same slides. Intestinal metaplasia and normal parietal cells were also often positive. In the study of 32 cases of benign stomach lesions, 2 out of 3 cases of atypical hyperplasia (ATP) and 3 out of 11 cases of stomach ulcer with regenerating epithelials were positive. Ras p21 was more dominantly expressed in the well-differentiated type of stomach cancer than the poorly differentiated type. Expression of ras p21, however, was not correlated either with the grades of cancer invasion or with the types of cancer infiltration.     

Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers

Sixteen clones (RASK-1 to -16) of murine monoclonal antibodies were raised against ras Mr 21,000 protein (p21). The p21 produced by Escherichia coli with inserted v-Ki-ras genes was used as immunogen. RASK-1 was found to be specific for Ki-ras p21, whereas RASK-2 to -16 reacted with the p21s of Ki-, N-, and Ha-ras genes in both enzyme-linked immunosorbent and immunoblotting assays. Binding inhibition assays with biotinylated monoclonal antibodies by enzyme-linked immunosorbent assay showed that the monoclonal antibodies of the 16 clones included those binding to several mutually distinct sites on p21. The expressions of ras p21 in human stomach and thyroid tissues were examined with RASK-3, which reacted with all the Ki-, N-, and Ha-ras p21s immunohistochemically by the avidin-biotin peroxidase complex method. Formalin-fixed, paraffin-embedded tissues of 101 cases of stomach cancer, 53 cases of noncancerous stomach, 74 cases of cancer of the thyroid, and 59 cases of noncancerous thyroid were analyzed. In both the stomach and thyroid, cancer cells expressed p21 predominantly. Cells of cases with various noncancerous disorders as well as certain types of normal cells were also p21 positive. These findings suggest that precaution is required in use of p21 as a cancer marker. Expression of p21 was noted in moderately to well-differentiated stomach cancer, intestinal metaplasia, and atypical hyperplasia. This finding suggests that the appearance of p21 in stomach cancer may be initiated before cytological transformation.     

癌基因产物ras P21在不同类型胃癌之表达

目的：探讨ras P21与胃癌生长速度之关系。方法：为在免疫组化的基础上探讨ras P21与胃癌生长速度之关系．作者设计了与过去文献不同的观察方法，不仅像过去文献那样观察胃癌之粘膜层之抗体表达，还分别观察粘膜下层、肌层、浆膜层及转移到局部淋巴或其他脏器癌细胞ras P21之表达，标本取自常观病理检查，均作H.E及ABC染色。结果：ras P21在胃癌细胞之检出率及反应强度在胃各层及局部淋巴转移的癌细胞中相似。早期胃癌ras P21检出率低于进展胃癌。结论：ras P21癌基因对胃癌之生长速度无明显影响。     

Human monoclonal macroglobulins with antibody activity

Assays for specific antigen-binding activity were performed on sera from 172 patients with monoclonal macroglobulinemia defined by immunofixation electrophoresis. The sera were collected between 1970 and 2002. Mean IgM level was 1,409 mg/dL with a range from 70 to 6,800. Cryoglobulins were identified in 15.3% (26/170 sera: 12 trace, five single component, and nine mixed IgM-IgG). Rheumatoid factor (RF) was detected in 19 of 151 (12.6%) samples with titers ranging from 1:80 to 1:327,680. Among the nine mixed IgM-IgG cryos, eight were RF-positive and six of six displayed positivity for hepatitis C virus. Cold agglutinins (CA) were present in 8.5% (10/117) of sera with anti-I titers between 1:512 and 1:65,536. IgM binding to a series of glycosaminoglycan oligosaccharides, glycolipids, and glycoprotein antigens was found in 75 samples (43%). IgM binding to antigens having known associations to polyneuropathies occurred in 20 patients (12%). Antinuclear antibody (ANA) was documented in 10.7% (18/169) of sera. Anti-DNA activity was absent in all samples tested. Sera from 71% of patients with monoclonal macroglobulinemia in this series exhibited binding to autoantigens. Some of these immune complexes resulted in clinically significant manifestations. Our results suggest that many monoclonal immunoglobulins may be functional antibodies rather than "paraproteins." Characterization of antigen-binding activities may provide insight into the pathogenesis of monoclonal gammopathies.     

Anti-tumor activity of an anti-endoglin monoclonal antibody is enhanced in immunocompetent mice

In the present study, we investigated the mechanisms by which anti-endoglin (EDG; CD105) monoclonal antibodies (mAbs) suppress angiogenesis and tumor growth. Antihuman EDG mAb SN6j specifically bound to murine endothelial cells and was internalized into the cells in vitro. SN6j effectively suppressed angiogenesis in mice in the Matrigel plug assay. We found that SN6j is more effective for tumor suppression in immunocompetent mice than in SCID mice. We hypothesized that T cell immunity is important for effective antitumor efficacy of SN6j in vivo. To test this hypothesis, we investigated effects of CpG oligodeoxynucleotides (ODN) and depletion of CD4(+) T cells and/or CD8(+) T cells on antitumor efficacy of SN6j in mice. Systemic (i.v.) administration of a relatively small dose (0.6 mug/g body weight/dose) of SN6j suppressed growth of established s.c. tumors of colon-26 in BALB/c mice and improved survival of the tumor-bearing mice. Addition of CpG ODN to SN6j synergistically enhanced antitumor efficacy of SN6j. In contrast, such enhancing effects of CpG ODN were not detected in SCID mice. Antitumor efficacy of SN6j in BALB/c mice was abrogated when CD4(+) T cells and/or CD8(+) T cells were depleted; effect of CD8(+) T cell depletion was stronger. Interestingly, CD4-depletion decreased tumor growth while CD8-depletion enhanced tumor growth in the absence of SN6j. SN6j induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner which indicates an additional mechanism of antiangiogenesis by SN6j. (c) 2008 Wiley-Liss, Inc.     

Detection of transforming ras proteins containing leucine at position 61 by a new mouse monoclonal antibody, ras(53-69)Leu61

A monoclonal antibody (mAb) was prepared after immunization of mice with a peptide that corresponds to amino acids 53 to 69 of a transforming ras protein. The amino acid sequence in this region is conserved among all members of the ras protooncogene family in rodent, rabbit, and human cells. The peptide used for immunization differs from the normal sequence by a single residue; Leu replaces Gln at a site corresponding to amino acid 61. A bacterial expression vector was constructed to synthesize H-ras transforming protein that contains this change (rasLeu61). In immunoblotting experiments, the affinity purified mAb, ras(53-69)Leu61, reacts specifically with the purified, bacterially produced rasLeu61 protein and does not react with bacterially produced normal H-ras protein. In immunoblotting experiments with cell lysates, the mAb recognizes the transforming protein in NIH3T3 cells transformed by the c-rasHLeu61 oncogene but fails to react with normal H-ras protein in the same cells or cells which produce 100 times more normal protein than NIH3T3. The mAb immunoprecipitates [35S]methionine-labeled H- and N-rasLeu61 proteins from transformed NIH3T3 cells under conditions in which the cells produce basal levels of the transforming protein, equivalent to the low amount of the normal protein ordinarily present in nontransformed NIH3T3 cells. The antibody fails to immunoprecipitate normal H-ras protein, even when present at high levels, or N-ras protein containing Lys as amino acid 61. Affinity purified mAb ras(53-69)Leu61 also recognizes the transforming ras protein specifically in immunohistochemical staining of tissue culture cells, and this staining is abolished by preincubating the antibody with the corresponding peptide. Staining was not observed with control NIH3T3 cells or cells that produce 100 times more normal H-ras protein than NIH3T3. However, in thin sections of normal human or rabbit skin the antibody reacted strongly with an unknown antigen, in cells of the basal layer of the epidermis, that is neither normal nor transforming ras protein. This new immunological reagent should be useful for the selective identification of Leu61 containing H-, K-, and N-ras transforming proteins in in vitro studies and analyses using rodent, rabbit, or human tissue culture cells. Its utility for direct staining of tissues may be limited to situations in which the presence of transforming protein can be verified by another method such as immunoblotting after gel electrophoresis.     

Potentiation of the reversal activity of SDZ PSC833 on multi-drug resistance by an anti-p-glycoprotein monoclonal antibody MRK-16

SDZ PSC833 (PSC833), an analogue of cyclosporines, is one of the most potent modulators of multi-drug resistance (MDR). We previously reported that MRK-16, an anti-P-glycoprotein MAb, enhanced MDR reversal activity of cyclosporin A (CsA) through inhibition of P-glycoprotein-mediated CsA transport. We have examined here whether MRK-16 can enhance MDR reversal activity of PSC833. We found that MRK-16 potentiated the MDR reversal activity of PSC833, and of CsA, in MDR sublines of human myelocytic leukemia K562 and human ovarian cancer A2780 cells. Like MRK-16 combined with CsA, MRK-16 enhanced the effect of a sub-optimum dose of PSC833 on vincristine accumulation in MDR cells. However, MRK-16 could not increase cellular accumulation of PSC833 in MDR tumor cells, yet it could increase cellular accumulation of CsA. P-glycoprotein could not transport PSC833 but could transport CsA. Our results indicate that MRK-16 potentiates the MDR reversal activity of both PSC833 and CsA, yet also suggest that the molecular mechanism of the potentiation differs between the two substances. © 1996 Wiley-Liss, Inc.     

Generation and characterization of an hKid-specific monoclonal antibody

Chromokinesins are chromosome-bound proteins during mitosis that play multiple important roles in chromosome segregation. The chromokinesin Kid has been shown to be involved in chromosome congression during mitosis and meiosis. Here we have generated a monoclonal antibody specific for the human chromokinesin hKid by immunizing BALB/c mice with a recombinant protein fragment corresponding to the C-terminal 250-amino acid residues of hKid. All five immunized mice responded excellently and gave high, nearly monospecific, antibody titers. One hybridoma, 8C12, was generated from spleen cells of a selected mouse, which recognized hKid on immunoblots and in immunofluorescence experiments. As hKid is an important regulator of chromosome segregation, this monoclonal antibody will be a useful tool for further analysis of this chromokinesin.     

Potent antitumor activity of an auristatin-conjugated, fully human monoclonal antibody to prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is the prototypic cell-surface marker of prostate cancer and provides an attractive target for monoclonal antibody (mAb) targeted therapies. In this study, a novel antibody-drug conjugate (ADC) was generated by linking a fully human PSMA mAb to monomethylauristatin E (MMAE), a potent inhibitor of tubulin polymerization. The PSMA ADC was evaluated for antitumor activity in vitro and in a mouse xenograft model of androgen-independent human prostate cancer. The PSMA ADC eliminated PSMA-expressing cells with picomolar potency and >700-fold selectivity in culture. When used to treat mice with established human C4-2 tumors, the PSMA ADC significantly improved median survival 9-fold relative to vehicle or isotype-matched ADC (P = 0.0018) without toxicity. Treatment effects were also manifest as significant (P = 0.0068) reduction in serum levels of prostate-specific antigen (PSA). Importantly, 40% of treated animals had no detectable tumor or measurable PSA at day 500 and could be considered cured. The findings support development of PSMA antibody-auristatin conjugates for therapy of prostate cancer.     

Inhibition of hemagglutinating activity by monoclonal antibody against Porphyromonas gingivalis 40-kDa outer membrane protein

Periodontitis is a chronic inflammatory disease of periodontal tissues that results in alveolar bone loss, and Porphyromonas gingivalis, which has a high hemagglutinating activity, has been implicated as an important pathogen in the development of periodontitis. This bacterium has a high hemagglutinating activity. We previously succeeded in gene cloning the 40-kDa outer membrane protein (OMP) from P. gingivalis 381. Although recombinant (r) 40-kDa OMP itself did not show hemagglutinating activity, its polymeric form, constructed with a cross-linking reagent, significantly expressed that activity. Furthermore, an affinity-purified antibody against r40-kDa OMP inhibited the hemagglutinating activity of P. gingivalis vesicles. In the present study, in order to clarify the pathological role of 40-kDa OMP and develop passive immunotherapy, we examined the inhibitory effect of monoclonal antibodies (MAbs) against r40-kDa OMP on the hemagglutinating activity of P. gingivalis vesicles. The MAbs reacted with r40-kDa OMP, the outer membrane fraction, vesicles, and P. gingivalis cell extracts, and significantly inhibited the hemagglutinating activities of the polymeric r40-kDa OMP as well as of P. gingivalis vesicles. These findings suggest that MAbs against 40-kDa OMP may be useful for the development of passive immunotherapy and for assessing treatment for periodontal diseases caused by P. gingivalis infection.     

抗Cripto单抗与羧胺三唑的联合抗肿瘤作用研究

目的探讨抗Cripto抗体（C13）对乳腺癌细胞的增殖抑制作用,以及抗Cripto抗体（C13）和羧胺三唑（CAI）合用是否有相加或协同作用,初步研究CAI与C13合用抑制肿瘤细胞增殖的作用机制。方法培养C13杂交瘤细胞,接种至SCID小鼠中,收集腹水,纯化后测定抗体效价。用不同浓度的C13抗体和CAI分别或共同处理MCF-7人乳腺癌细胞和66c14小鼠乳腺癌细胞。利用[^3H]-胸腺嘧啶掺入法分析CAI与C13对细胞DNA合成的影响。对细胞进行碘化丙啶染色,利用流式细胞仪评价两种药物对细胞凋亡的影响。结果在上述两种细胞系中,C13抗体无论是单用还是与CAI合用对肿瘤细胞的生长均有不同程度的抑制作用,且该作用呈剂量依赖性。流式细胞分析结果显示,C13单独或与CAI一同处理MCF-7细胞和66c14细胞后,以上细胞亚二倍体DNA的细胞比例升高,药物合用后该作用将进一步增加。结论针对Cripto17肽的抗体C13与CAI联用可以增强前者对乳腺癌细胞的抑制效应,这一作用可能与诱导细胞凋亡有关。     

Production of monoclonal antibody inhibiting dipeptidylaminopeptidase IV activity of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic bacterial species implicated as an important pathogen in the development of adult periodontitis. We previously cloned a gene encoding dipeptydilaminopeptidase IV (DAPIV) from P. gingivalis. In the present study, for immunological diagnosis and development of passive immunization, we produced a mouse monoclonal antibody (MAb) capable of inhibiting the DAPIV activity of P. gingivalis using highly purified recombinant DAPIV as an immunogen. The constructed MAb, designated as MAb-Pg-DAP-1, significantly inhibited DAPIV activity in P. gingivalis, as well as slightly inhibited that in other gram-negative bacteria such as Porphyromonas endodontalis and Prevotella loesheii, whereas no inhibition was seen in the gram-positive bacteria Streptococcus mutans and Actinomyces viscosus. Furthermore, the MAb did not inhibit DAPIV enzyme activity in human serum. This novel MAb may be useful for the development of immunological diagnosis capability and in passive immunization.     

Inhibition of hepatocellular carcinoma cell growth by an anti-insulin-like growth factor-I receptor monoclonal antibody

Hepatocellular carcinoma (HCC) overexpresses insulin-like growth factor-I receptor (IGF-IR), as compared with normal hepatocytes. Since IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for treating HCC. Here, we have generated a murine anti-IGF-1R antibody, 4F2, that recognizes the IGF-IRα subunit and blocks in?vitro IGF-I and IGF-II-induced cell proliferation of SMMC-7721 and Bel-7402 HCC cell lines. 4F2 can inhibit IGF-IR autophosphorylation, IRS-1 phosphorylation and the activation of the major downstream signaling molecules AKT and mitogen-activated protein kinase. Additionally, we observed a moderate increase in apoptosis as demonstrated by detection of changes in the expression of the pro-apoptotic and anti-apoptotic proteins Bax and Bcl-2 after 4F2 treatment. Combined treatment with 4F2 and doxorubicin was more effective in reducing cell proliferation and promoting apoptosis than either agent alone. These data support that therapeutic anti-IGF-IR antibodies are potential new agents for treating HCC.     

Histologic demonstration of antigens reactive with anti-p21 ras monoclonal antibody (RAP-5) in human stomach cancers

The immunohistochemical reactivity of RAP-5, a monoclonal antibody (MoAb) raised against a synthetic peptide corresponding to positions 10-17 of the ras gene product from T24 bladder carcinoma, was studied in 96 surgically resected stomach cancers of humans. The cytoplasm of cancer cells in 65 cases (68%) was positively stained with MoAb RAP-5, although the staining was heterogeneous among cancer cells. There was no definite correlation between depth of tumor invasion and reactivity to MoAb RAP-5. Cancer cells of poorly differentiated tumors showed a tendency to react less frequently and less intensely to MoAb RAP-5. In nontumorous gastric mucosa, parietal cells and some portions of intestinal metaplasia were stained with MoAb RAP-5. These findings suggest an increased expression of the ras gene product (p21) in about two-thirds of gastric adenocarcinomas and in some nonneoplastic gastric epithelial cells.     

Stimulation of exocytotic degranulation by microinjection of the ras oncogene protein into rat mast cells

To investigate the possible role of ras proteins in the secretory process, we have microinjected the proto oncogenic and oncogenic forms of the human H-ras protein into rat peritoneal mast cells. Mast cells are secretory cells which, upon appropriate stimulus, liberate histamine and other mediators of the acute inflammatory reaction by exocytotic degranulation. We report here that microinjection of the ras oncogene protein into mast cells induces exocytotic degranulation. In contrast, microinjection of similar amounts of the proto-oncogenic protein has little apparent effect on mast cells. Degranulation induced by injection of the ras oncogene protein occurs in the absence of an external stimulus and requires the presence of external calcium. The ultrastructural features of exocytotic degranulation in mast cells injected with the ras oncogene protein are similar to those seen when mast cells are activated by soluble ligands. Our results suggest that ras proteins may be involved, possibly as regulatory elements, in cellular functions that control exocytosis.     

Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia

CD19 is a pan B-cell surface receptor expressed from pro-B-cell development until its down-regulation during terminal differentiation into plasma cells. CD19 represents an attractive immunotherapy target for cancers of lymphoid origin due to its high expression levels on the vast majority of non-Hodgkin's lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcgamma receptors on immune cells and thus increase Fc-mediated effector functions. In vitro, XmAb5574 enhanced antibody-dependent cell-mediated cytotoxicity 100-fold to 1,000-fold relative to an anti-CD19 IgG1 analogue against a broad range of B-lymphoma and leukemia cell lines. Furthermore, XmAb5574 conferred antibody-dependent cell-mediated cytotoxicity against patient-derived acute lymphoblastic leukemia and mantle cell lymphoma cells, whereas the IgG1 analogue was inactive. XmAb5574 also increased antibody-dependent cellular phagocytosis and apoptosis. In vivo, XmAb5574 significantly inhibited lymphoma growth in prophylactic and established mouse xenograft models, and showed more potent antitumor activity than its IgG1 analogue. Comparisons with a variant incapable of Fcgamma receptor binding showed that engagement of these receptors is critical for optimal antitumor efficacy. These results suggest that XmAb5574 exhibits potent tumor cytotoxicity via direct and indirect effector functions and thus warrants clinical evaluation as an immunotherapeutic for CD19(+) hematologic malignancies.