Armed and targeted measles virus for chemovirotherapy of pancreatic cancer.

No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.

The prostate stem cell antigen represents a novel glioma-associated antigen.

Gliomas of WHO grades III-IV are malignant brain tumors mostly resistant to conventional therapies. Therefore, novel strategies for the treatment of gliomas are warranted. Although immunotherapy is gaining increased attention for the treatment of malignant gliomas and in particular of glioblastoma multiforme (GBM), this approach requires the identification of appropriate antigens. Our aim was to investigate the expression of the prostate stem cell antigen (PSCA), a highly N-glycosylated phosphatidylinositol (GPI)-anchored cell surface protein, in gliomas of different WHO grades in order to evaluate its potential as a diagnostic marker and as a target for immunotherapy. Tumor specimens and controls were assessed by quantitative RT-PCR, Western blotting and immunohistochemistry. The samples investigated in the study consisted of 210 human glial tumors, among which 31 were oligodendrogliomas, 9 ependymomas and 170 were astrocytomas (including 134 glioblastomas). PSCA was absent in normal brain tissue, but was detected in WHO grade III-IV gliomas. Weak PSCA protein expression was also recognized in some WHO grade I and WHO grade II tumors. The difference between WHO grade I-II tumors and WHO grade III-IV tumors was statistically significant (p<0.001). Our results suggest that increased PSCA expression levels are linked to gliomas of WHO grades III and IV, and may represent a suitable additional target for immunotherapy of gliomas.

Detection of autoantibodies to tumour-associated antigens in sera of patients with systemic autoimmunity using a novel protein microblot array.

It is well known that sera of patients with systemic autoimmunity contain autoantibodies to nuclear antigens. It is also known that patients with systemic autoimmunity have an increased risk for the development of tumours. Interestingly, tumour patients frequently develop autoantibodies and there is a growing list of potential tumour-associated antigens. It is, however, not known whether or not patients with systemic autoimmunity also develop antibodies to tumour-associated antigens. Here we describe the development of a novel multiprotein array allowing us to screen for autoantibodies to 30 different tumour-associated antigens in parallel. Using this novel assay, we found that the frequency of autoantibodies to the selected tumour-associated antigens is increased between 2- and 14-fold in patients with systemic autoimmunity compared with an age-matched control group.

Bortezomib significantly impairs the immunostimulatory capacity of human myeloid blood dendritic cells.

Bortezomib is a potent drug for the treatment of multiple myeloma. Its anti-tumor activity is mediated by proteasome inhibition leading to decreased cell proliferation and induction of apoptosis. However, an unimpaired proteasomal function plays a crucial role for the induction of anti-tumor immunity by dendritic cells (DCs), which are currently used for therapeutic vaccination against various tumors including myeloma. In the present study, we investigated the impact of bortezomib on the immunostimulatory capacity of 6-sulfo LacNAc (slan) DCs, which represent a major subset of human blood DCs. We demonstrated that this proteasome inhibitor efficiently impairs the spontaneous in vitro maturation of slanDCs and the release of tumor necrosis factor (TNF)-alpha as well as interleukin (IL)-12 upon lipopolysaccharide (LPS) stimulation. Functional data revealed that bortezomib profoundly inhibits slanDC-induced proliferation and differentiation of CD4(+) T cells. In addition, the capacity of slanDCs to promote interferon-gamma secretion and tumor-directed cytotoxicity of natural killer (NK) cells is markedly impaired by bortezomib. These results provide evidence that bortezomib significantly reduces the ability of native human blood DCs to regulate innate and adaptive anti-tumor immunity and may have implications for the design of therapeutic strategies combining DC vaccination and bortezomib treatment.

Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy.

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A(*)0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

Inhibition of malignant glioma cell growth by a survivin mutant retrovirus.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor that is resistant to conventional radiotherapy and chemotherapy. The median survival time of patients with GBM has remained less than 2 years despite concerted efforts to improve therapy. As a new approach to treat GBM we generated retroviral particles encoding mutant survivin for transduction of glioma cells. We demonstrate here that retroviral overexpression of a nonphosphorylatable Thr-34 --> Ala mutant of survivin (survivinT34A), in the glioma cell lines U373 and H4 resulted in a marked increase in the percentage of cells bearing multiple nuclei, which was accompanied by significantly decreased cell proliferation, and in greater numbers of cells with hypodiploid DNA content. Administration of the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl-ketone did not reduce the cell death rate. Yet increased nuclear translocation of apoptosis-inducing factor (AIF) was observed in cells transduced with survivinT34A, indicating caspase-independent cell death. Transduction of retroviral vectors encoding wild-type survivin also led to the appearance of multinuclear cells. In contrast to mutant survivin, overexpressed wild-type survivin did not increase the cell death rate and no enhanced nuclear AIF translocation was observed. We suggest that retroviral vectors delivering mutant survivinT34A might be employed for the treatment of glioblastoma.

Identification of an HLA-A*0201-restricted T-cell epitope derived from the prostate cancer-associated protein prostein.

The development of T-cell-based immunotherapies of cancer largely depends on the availability of tumour-associated antigens capable of eliciting tumour-directed cytotoxic T-cell responses. In prostate cancer, the number of antigens defined as suitable targets of cytotoxic T lymphocytes (CTLs) is still limited. Recently, prostein was identified as a transmembrane protein that is highly restricted to prostate tissues. In our study, prostein transcripts were found to be abundant in both malignant and nonmalignant prostate tissue samples. To identify immunogenic CD8+ T-cell epitopes, human leucocyte antigen-A(*)0201-binding peptides were selected from the amino-acid sequence of prostein and were used for the in vitro stimulation of CD8+ T lymphocytes. Specific CTLs were raised against the prostein-derived peptide CLAAGITYV that were capable of lysing prostate cancer cells, indicating that this peptide is naturally generated by tumour cells. Our data suggest that prostein is a suitable candidate to be included in a T-cell-based immunotherapy of prostate cancer.

Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34(+) cord blood-derived dendritic cells.

Differentiation of genetically modified CD34(+) hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34(+) cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.

An in vitro vitellogenin bioassay for oestrogenic substances in the medaka (Oryzias latipes).

We developed an in vitro bioassay for oestrogenic compounds based on vitellogenin as a biomarker. To quantify the induction of vitellogenin in medaka (Oryzias latipes) we established a sandwich enzyme-linked immunosorbent assay (ELISA). This ELISA was carried out with two different monoclonal antibodies specific for the yolk precursor protein vitellogenin and the high molecular weight yolk proteins (lipovitellin) of medaka. Purified lipovitellin of medaka was used as a standard for the ELISA and could be quantified down to a concentration of 9 ng/ml. The calibration curve was linear up to at least 600 ng/ml and the intra-assay coefficient of variance was 3.2%. The application of the sandwich ELISA was tested using blood samples of males and females as well as primary hepatocyte cultures of medaka. Vitellogenin synthesis in cultured liver cells of males was induced by 1 and 100 nM of the xenoestrogen 17alpha-ethynylestradiol (EE(2)). The first production of vitellogenin was detected 6 days after hormone application. In contrast, isolated liver cells of sexually mature females, which were treated in the same manner, showed vitellogenin expression from the beginning of cultivation and its synthesis increased and remained high at 100 nM EE(2). However, the induction of vitellogenin synthesis in male hepatocytes in vitro could be maintained for at least one month and this indicated viable and differentiated liver cells. The hepatocyte cultures of male medaka in combination with the sandwich ELISA were shown to be a suitable tool to detect and quantify oestrogenic activity of chemicals.

Quantification of antigen-reactive T cells by a modified ELISPOT assay based on freshly isolated blood dendritic cells.

The enzyme-linked immunospot (ELISPOT) assay has become a widely employed method for quantification of antigen-reactive T lymphocytes. In recent years, various types of antigen-presenting cells (APCs) have been tested as stimulator cells in ELISPOT protocols to achieve a highly sensitive and rapid assay which is not impaired by a marked nonspecific cytokine release. However, the currently available APCs still have disadvantages, such as significant background reactivities, limited sensitivity, and time-consuming preparation procedures. Recently, we succeeded in defining a novel subpopulation of circulating dendritic cells (DCs) that can easily be prepared from human blood. These M-DC8+ DCs proved to be very effective in the induction of antigen-specific T cell responses. In the present study we provide evidence that M-DC8+ DCs are particularly well suited as APCs for the detection of antigen-specific CD8+ T cells after challenge with viral or tumor peptides in ELISPOT assays. In addition, protein-loaded M-DC8+ DCs proved to be quite efficient in the presentation of MHC class II-bound peptides, thus allowing the determination of frequencies of antigen-reactive CD4+ T cells. The use of M-DC8+ DCs as stimulator cells can improve the ELISPOT assay by combining high sensitivity, rapidity, and low background reactivity.

Increased survivin transcript levels: an independent negative predictor of survival in soft tissue sarcoma patients.

Survivin, a recently identified inhibitor of apoptosis protein (IAP), is expressed in diverse embryonic tissues and in various human cancers. We have investigated the quantitative expression of survivin mRNA by a sensitive TaqMan-based RT-PCR assay in tissue samples from 94 patients with soft tissue sarcomas (STS). Survivin transcript levels were measured and normalized to GAPDH transcripts. By using a multivariate Cox regression analysis, we found an inverse correlation between the level of survivin mRNA (ratio >2 zmol survivin/amol GAPDH) and the rate of overall survival (p = 0.009, RR = 2.7). Survivin transcript variants as detected by qualitative RT-PCR analysis were revealed in 36 of 56 STS patients (64%). Only survivin DeltaEx3 and/or full-length survivin variants but not survivin 2B were identified. Our results suggest that a higher level of survivin mRNA is an independent predictor of survival for STS patients.

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.

Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.

Generation of survivin-specific CD8+ T effector cells by dendritic cells pulsed with protein or selected peptides.

The identification of tumor-associated antigens recognized by CD8+ cytotoxic T cells paved the way to new concepts in adjuvant anticancer therapy. However, the number of tumor-associated proteins found to be expressed in the majority of human cancers is still rather limited. Recently, the newly identified apoptosis inhibitor protein survivin has been recognized as a widely occurring tumor-associated protein. In the present study, we demonstrate that survivin is capable of inducing specific CD8+ effector T cells in vitro. T cells from healthy donors were subjected to several cycles of stimulation by autologous dendritic cells (DCs) pulsed with soluble recombinant survivin protein. Activation of CD8+ cytotoxic T cells by survivin-derived peptides cross-presented by DCs was demonstrated by lysis of autologous survivin-expressing B cell transfectants. Using a peptide-motif scoring system, two survivin peptides (ELTLGE-FLKL and TLPPAWQPFL) were predicted and proved to bind to the HLA-A*0201 molecule. Both peptides were shown to induce CD8+ effector T cells when presented on DCs; one peptide could be verified to result from natural intracellular processing of survivin. These findings recommend survivin as a new and widely applicable target for protein- and peptide-based immunotherapy of tumors.

Expansion of CD60 helper lymphocytes detected in peripheral lymphocytes of HIV-1 infected individuals is not paralleled in lymph nodes.

BACKGROUND AND OBJECTIVE: A significant expansion of CD8 cells with capability of Th2 type helper function had been observed in hemophiliacs with HIV infection. These cells were characterised by the surface co-expression of CD8 and CD60 antigen. Our objective was to investigate this lymphocyte subset in relation to other subsets in homosexuals and drug users in two compartments: blood and lymph nodes. Blood and lymph nodes from not HIV-infected persons served as control. RESULTS: CD8+CD60+ cells were expanded in perpheral blood of HIV - infected patients as compared to age matched controls (10.0 versus 4.1%, p <0.05). This difference was not observed in lymph node cell suspensions (6.2 vs. 4.3% of all lymph node cells; p = n.s.). The CD 4/CD8 ratio was significantly less impaired in lymph nodes than in blood (2.27 vs. 0.83; p <0.05). Cytotoxic T cells were more abundant in the lymph nodes of patients with early stage HIV disease when compared two late stage patients (4.3 vs 2.1%; p <0.05). Immunohistochemistry on frozen lymph node cuts showed presence of CD60 cells mainly in the interfollicular and paracortical area. In 3 of 10 HIV infected patients these cells were also found in the germinal centers. In controls no CD60 cells were detected in the follicles. Numbers and percentages of CD60 cellls and CD8+CD60+ cells in blood and in lymph nodes did not correlate with HIV - stage, CD4 count or plasma viral load. No correlation with lymph node viral load was seen. CONCLUSIONS: Our data confirm that like in hemophiliacs expansion of CD8+CD60+ is also found in the blood of other HIV risk groups and seems not to be specific for hemophiliacs. However, the higher percentage in peripheral blood is not paralleled in lymph nodes. Redistribution phenomena seem to be the most plausible explanation. According to these data, a major impact of CD8+CD60+ cells in the immunopathogenesis of HIV infection does not seem likely.

Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients.

Antibody reactivity against survivin, a recently identified tumor-associated protein, was determined in sera from patients with lung (n = 51) or colorectal cancer (n = 49). The same collection of sera was tested for the presence of antibodies against p53. Eleven sera from lung cancer patients and four sera from colorectal cancer patients reacted with purified recombinant survivin in an ELISA (21.6% and 8.2%, respectively), whereas four sera from lung cancer patients and nine sera from colorectal cancer patients contained anti-p53 antibodies (7.8% and 18.4%, respectively). The increase in prevalence when anti-survivin and anti-p53 antibodies were determined in parallel was statistically significant (29.4% versus 7.8%, P = 0.005 in lung cancer population; 26.6% versus 8.2%, P = 0.015 in colorectal cancer population). The high prevalence of anti-survivin antibodies makes these antibodies an attractive novel marker for the diagnosis of lung and colorectal cancer, particularly in patients lacking anti-p53 antibodies.