DNA-based tumor vaccines.

Tumor vaccination strategies have been increased over the past years. This increase began with the identification of tumor antigens recognized by the immune system. Better understanding of the immune system and increasing knowledge about the antigen presentation process and the role of dendritic cells have opened new therapeutic possibilities. DNA vaccines, already successfully used against viral antigens and covering a broad repertoire of epitopes, might also be of advantage in tumor immunotherapy. Design and selection of vectors are of considerable importance for the vaccination. There are three major types of DNA-based recombinant cancer vaccines: DNA from tumor antigens can be used 1) to modify dendritic cells, 2) as 'naked' DNA-vaccine or 3) to construct recombinant viral vaccines. The current status in this field is shown and different vaccination strategies are discussed.

Efficient cryopreservation of dendritic cells transfected with cDNA of a tumour antigen for clinical application.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA-DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials.

Mucin gene (MUC1) transfer into human dendritic cells by cationic liposomes and recombinant adenovirus.

BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC.

Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1).

Naked DNA is an attractive tool for vaccination studies. We have studied naked DNA vaccination against the human tumor antigen mucin, encoded by the gene MUC1. C57/BL6 mice were immunized twice, on day 1 and day 10. with plasmid pCI-MUC1, intramuscularly. Five days after the last immunization tumor challenge experiments were performed using the tumor cell line MC38, expressing human MUC1. In 85% of mice immunized with the mucin plasmid tumor growth inhibition was observed, whereas control mice developed tumors. Re-tumor challenge after three months revealed no tumor growth in mice immunized with the mucin plasmid. These encouraging results, showing long-term protection against tumor growth, indicate the potential usefulness of naked DNA vaccination for clinical immunotherapy.

Generation of an immortalized human CD4+ T cell clone inhibiting tumor growth in mice.

Tumor antigen-specific T cell clones represent a useful tool in tumor immunology; however, their long-term culture is limited. To generate an immortalized cytotoxic T cell clone against the human tumor antigen mucin, we exposed a previously generated T cell culture to Herpesvirus saimiri. We obtained an immortalized human CD4+ T cell clone, termed SITAM. Clonality of these cells was shown by analysis of the alpha/beta-T cell receptor (TCR) repertoire. Cytolytic activity was demonstrated against several mucin-expressing tumor cell lines and could not be detected against non-mucin-expressing cells. SITAM cells maintained their features stably for 2 years. Furthermore, growth of the tumor cell line Capan-2 in NOD/SCID mice was inhibited when SITAM cells were coinjected subcutaneously with tumor cells. SITAM cells provide an unlimited source of clonal T cells for analysis of tumor recognition and may be of help in TCR-targeted immunotherapy.

Up-regulation of telomerase activity in Herpesvirus saimiri immortalized human T-lymphocytes.

Human T-lymphocytes can be transformed to unlimited growth by Herpesvirus saimiri (HVS). We studied the telomerase activity of a recently established HVS immortalized human CD4 T cell clone in comparison to peripheral blood lymphocytes (PBL) and unstimulated or phytohemagglutinin (PHA)-stimulated CD4 T-cells by a Telomeric Repeat Amplification-Protocol (TRAP) -Assay. Telomerase activity in PHA-stimulated CD4 T-cells was seven-fold and in HVS-infected CD4 T-cells 14-fold higher than in untreated CD4 T-cells. The HVS immortalized T-cell clone provides a useful tool for studying the regulation of telomerase activity during carcinogenesis and for testing of telomerase-inhibitory drugs.

Tumor-specific targeting of a cell line with natural killer cell activity by asialoglycoprotein receptor gene transfer.

Targeting of immunological effector cells to tumor cells could be an efficient strategy of adoptive immunotherapy. The success of this strategy depends on the specificity of the effector cells and their availability in sufficient numbers. The aim of this study was to target the human natural killer cell line YT specifically to tumor cells. The cell line was modified by transfection with the cDNA of the human asialoglycoprotein receptor (ASGPR). This C-type lectin recognizes carbohydrates containing terminal galactosyl (Gal) residues, including the beta1-Gal bearing Thomsen-Friedenreich (TF) antigen, which is found on tumor cells. Binding assays revealed that the ASGPR-gene-transfected YT cell line binds significantly higher to tested target tumor cell lines than the mock-transfected control cells. Cytolytic activity against the tumor cell lines Raji, Jurkat and the TF-positive KG1 subline was increased. Genetic modification of YT cells could provide a useful tool for tumor targeting in immunotherapy.

Binding of tumor antigen mucin (MUC1) derived peptides to the heat shock protein DnaK.

The human epithelial mucin encoded by the gene MUC1 is a tumor-associated antigen expressed on breast, pancreatic, colon and ovarian cancer cells recognized by cytotoxic T-cells and antibodies. Underglycosylated as well as glycosylated mucin-peptide epitopes are promising targets for vaccination against cancer. Heat shock proteins of 70 kDa (HSP70), also highly expressed in tumor cells, can function as chaperones for peptides and proteins and are involved in antigen processing. The involvement of HSP70 molecules in mucin antigen binding, processing and presentation has not yet been examined. Here we present first results concerning the relative binding affinities of various mucin-derived peptides to the bacterial 70 kDa heat shock protein DnaK. Interestingly, longer mucin peptides reveal a higher affinity to DnaK than short peptides. The non-glycosylated mucin-derived peptides of 5-8 amino acids length were able to compete with a high affinity (unrelated) reference peptide at millimolar concentrations. Glycosylation of the investigated short peptides lowers their binding affinity to DnaK, depending on the position of the carbohydrate. The binding affinity is not influenced by free charges at unprotected ends. The peptide (MUC1)5 consisting of five repeating units has an affinity enhanced by a factor of three as compared to the peptide with only one repeating unit. Mucin-peptide-HSP-complexes could be the basis of developing new kinds of tumor vaccines.

Wilms' tumor gene (WT1) expression in lung cancer, colon cancer and glioblastoma cell lines compared to freshly isolated tumor specimens.

The Wilms' tumor gene (WT1) encodes a transcriptional regulator involved in growth and differentiation of various tissue types. A continuous over-expression of WT1 was found in leukemic blasts, thus suggesting an oncogenic function. Solid cancer entities have also been described as expressing WT1. We systematically analyzed WT1 expression in small-cell and non-small-cell lung cancer, colon cancer and glioblastoma patients and in the respective tumor cell lines. Using reverse transcription/polymerase chain reaction, we found WT1 expression in glioblastoma (5 of 8), lung (5 of 11), and colon cancer (5 of 15) cell lines. While WT1 was expressed in only 1 of 12 lung cancer and 1 of 5 glioblastoma specimens, it was not detected in colon cancer or macroscopically tumor-free colon and lung tissue. In addition, HT29 colon cancer cells showed a loss of WT1 expression when grown to confluence or induced to differentiate by sodium butyrate. From this evidence, testing for WT1 expression is not clinically relevant for colon cancer, lung cancer, or glioblastoma patients. WT1 expression in cancer cell lines can probably be attributed to optimized in vitro growth conditions.

Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalized B-cells to generate tumor antigen specific cytotoxic T cells.

BACKGROUND: EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses. Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro. These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV. In addition, mini-EBVs can be modified in E. coli to alter their genetic composition or adopt new genes. METHODS: We constructed a mini-EBV plasmid carrying an expression cassette for the human tumor antigen mucin encoded by the gene MUC-1. Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro. These B-cells were analyzed by FACS analyses for the expression of mucin and co-stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors. RESULTS: Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV. These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation. Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established. CONCLUSIONS: Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines.

[Reduction of radiation exposure for patient and investigator in interventional radiography]. / Reduktion der Strahlenexposition in der interventionellen Angiographie für Patient und Untersucher.

PURPOSE: In a retrospective analysis of vascular interventional procedures, relations between parameters of the examination and radiation exposure of patient and medical personnel are examined. MATERIAL AND METHOD: 1208 vascular interventional procedures are evaluated. Interventional procedures are divided into three groups: percutaneous transluminal angioplasty, implantation of a stent, thrombolysis. RESULTS: Mean values of the radiation dose of patient and radiology personnel are reported for these examinations. The mean value of the radiation dose of the physician was 7 microSv (maximum 24 microSv), that of the patient 1548 cGy.cm2 (maximum 8485 cGy.cm2). CONCLUSION: The quantity of X-rays to the patient may be lowered by using pulsed fluoroscopy and by reducing the number of radiographs. Reduction of the number of radiographs may be achieved by using the last-image hold and the road mapping mode. The operator's dose can be decreased by using additional radiation protection systems like a MAVIG-radiation protection wall. The radiation dose reduction was 61% for the physician and 17% for the patient.

A survivor of breast cancer with immunity to MUC-1 mucin, and lactational mastitis.

The human mucin, MUC-1, is a transmembrane glycoprotein that is produced by both normal an malignant epithelium. The MUC-1 produced by malignant epithelium is underglycosylated, which leads to the expression by tumors of novel T and B cell epitopes on the mucin polypeptide core. Similar underglycosylation occurs in the lactating breast. In this report, we describe a long-term survivor of breast cancer whose tumor strongly expressed the T- and B-cell-stimulatory epitopes. Five years after presenting with the tumor, the patient had her first pregnancy, at which time she developed fulminant lymphocytic mastitis. We demonstrate that the lactating breast produced mucin expressing the same "tumor-specific" epitopes as the original cancer. The patient had circulating anti-mucin antibodies of both the IgM and IgG isotypes (these are not found in normal controls), and mucin-specific cytotoxic T lymphocytes in the peripheral blood. Limiting-dilution analysis for mucin-specific cytotoxic T lymphocytes in three different experiments yielded frequencies of 1 in 3086, 1 in 673, and 1 in 583, compared to approximately 1 in 10(6) in normal controls. The patient remains clinically free of carcinoma after 5 additional years of follow-up. We propose that the original tumor primed the patient's immune response against the mucin epitopes, and that the re-expression of these epitopes on the lactating breast evoked a secondary immune response. It is tempting to speculate that the vigor of her anti-mucin immunity may have helped protect this patient against recurrent tumor.

Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

MUC-1 epithelial tumor mucin-based immunity and cancer vaccines.

Many obstacles still stand in the way to eliciting an effective immune response against cancer, even though several antigens and antigenic peptides have been identified as potential tumor targets. All of them, including the MUC-1 mucin, share the caveat of being normal cellular proteins. Unlike all the others, however, MUC-1 expressed on tumors can still be considered a truly tumor-specific antigen. Its expression on normal cells is hidden from the immune system, and its aberrant glycosylation on tumors creates new epitopes recognized by the immune system. Moreover, all other tumor targets identified so far are MHC-restricted peptides that can only be recognized by patients who carry a specific HLA type, or on tumors which continue to express particular HLA alleles. MUC-1 is powerfully different. Recognized as a native molecule independent of MHC, it is a universal immunogen and a universal target, and if made effectively immunogenic, it would be expected to elicit immune responses in all patients, and against numerous MUC-1 expressing human tumors. It may, in fact, be the extraordinary solution to an extraordinary problem of cancer immunity and immunotherapy.

Humoral immunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast, pancreatic, and colon cancer patients.

Using synthetic peptides 60,80, and 105 residues long, corresponding to 3, 4, and 5.25 tandem repeats of human mucin MUC-1 protein core, as antigens in a solid-phase enzyme-linked immunosorbent assay, we screened sera from 24 breast cancer patients, 10 colon cancer patients, and 12 pancreatic cancer patients, at various stages of disease, for the presence of mucin-specific antibodies. The 105-residue peptide was superior in allowing detection of high levels of anti-mucin antibodies in 10.9% of sera in each cancer group. Another 4.3% showed intermediate reactivity. Lower levels of detection were achieved with the 80-residue peptide, and no specific reactivity was detectable with the 60-residue peptide. Anti-mucin antibodies were previously undetectable when this assay was performed with purified whole mucin or short synthetic peptides. The presence or absence of antibody did not correlate with the levels of circulating mucin or stage of disease. One highly reactive serum sample was used to identify more precisely the epitope on the long synthetic peptide to which the reactivity was directed. The reactivity of this serum specific for the 105-residue peptide was blocked by a 9-residue peptide from the NH2-terminal region of the 20-residue tandem repeat containing the previously identified immunogenic epitope APDTRP. Another 9-residue mucin peptide, from the COOH-terminal region of the tandem repeat which does not contain the APDTRP epitope, had no effect. All the mucin-specific reactivity was found to be of the IgM isotype, indicating a helper T-cell-independent response, unusual for an antibody against a peptide epitope, but not unexpected for tandemly repeated epitopes.

Increased excretion of endogenous urinary leukotriene E4 in extrahepatic cholestasis.

The cysteinyl leukotrienes LTC4, LTD4 and LTE4 are potent lipid mediators eliminated from the blood circulation mainly due to uptake by the liver and the kidneys. In man hepatobiliary elimination of cysteinyl leukotrienes predominates over renal excretion. In the present study, the urine from patients with extrahepatic cholestasis (n = 25) and age- and sex-matched healthy control subjects (n = 25) was analyzed for endogenous LTE4, the predominant metabolite of LTC4 excreted into urine. LTE4 was separated by reversed-phase high-performance liquid chromatography and subsequently quantified by enzyme immunoassay. Healthy subjects excreted a median concentration of 14 nmol LTE4/mol creatinine (range 5-24 nmol/mol creatinine). Its median concentration increased significantly to more than 5-fold higher levels to 74 nmol LTE4/mol creatinine (range 52-93 nmol/mol creatinine) in patients with extrahepatic cholestasis (P < 0.01). These results indicate that extrahepatic cholestasis leads to a compensatory diversion of cysteinyl leukotriene elimination to the kidney with subsequent increased excretion of LTE4 into urine.