Serguei Zoubak

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer : A phase I/II clinical trial

Introduction and Objectives: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor–specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by ‘naked’ DNA immunization or by injecting replication–deficient viral vectors that carry the tumor–specific DNA. Using the prostate– specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections.Methods: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty–six patients with prostate cancer were entered into a phase I/II toxicity–dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 μg and of recombinant virus at 5×108 PFUs per application were used.Results and Conclusion: No immediate or long–term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed–type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM–CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM–CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Fas and Fas Ligand Expression on Human Peripheral Blood Leukocytes

Objectives: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes. Methods: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears, Western blotting. Results: Granulocytes and monocytes express a low level of Fas receptor, but no Fas-L. These cells, as well as NK cells, contain presynthesized depots of Fas-L which they express following activation by brief storage (60 min) at room temperature or during separation from whole blood. Such activation also leads to Fas receptor upregulation. NK cells do not express Fas receptor. Once expressed on blood leukocytes, fully functional Fas-L can be released from the membrane and can be detected in plasma-free cell supernatants. Conclusion: Human peripheral blood granulocytes, monocytes and NK cells contain intracellular presynthesized Fas-L which they readily express following blood anticoagulation, blood storage or cell separation. Soluble Fas-L is released from those cells and can be detected in protein-free supernatants by immunoblotting.

Immune responses against PSMA after gene-based vaccination for immunotherapy – A: results from immunizations in animals

Two plasmid DNA vaccines, encoding either products that are retained in the cytosol and degraded in the proteasome (tVacs; hPSMAt), or secreted proteins (sVacs; hPSMAs) were evaluated for stimulation of cytotoxic cell or antibody responses. Immunization with both vectors led to generation of cell cytotoxicity providing granulocyte-macrophage colony-stimulating factor was administered with the vaccine. Spleen cells from animals immunized with hPSMAt demonstrated stronger cytotoxicity to the target cells. Priming with a vector that encoded a xenogeneic protein (hPSMAt; ‘xenogeneic’ construct) and boosting with a vector that encoded an autologous protein (rPSMAt; ‘autologous’ construct) gave the best protection against tumor challenge. Immunization with tVacs did not lead to formation of antibodies to the target protein as detected by Western blot or ELISA, while immunization with sVacs or with the protein did. Antibodies were of mixed Th1–Th2 isotype. Priming with tVacs and boosting with protein also resulted in antibody formation, but in this case the antibodies were from the cytotoxic, Th1 isotype. The best strategy to obtain a strong cellular cytotoxic response, therefore, seems to be gene-based vaccinations with tVacs, priming with the ‘xenogeneic’ and boosting with the ‘autologous’ constructs. When cytotoxic antibody production is the goal, priming should be performed with the tVacs while boosting with the protein.

Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes

The ability of two plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses is demonstrated. The first vaccine (truncated; tPSMA) encodes for only the extracellular domain of prostate-specific membrane antigen (PSMA). The product, expressed following transfection with this vector, is retained in the cytosol and degraded by the proteasomes. For the “secreted” (sPMSA) vaccine, a signal peptide sequence is added to the expression cassette and the expressed protein is glycosylated and directed to the secretory pathway. Monocyte-derived dendritic cells (DCs) are transiently transfected with either sPSMA or tPSMA plasmids. The DCs are then used to activate autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs or with peptide-pulsed monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tPSMA DCs and sPSMA DCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted toward one of the four PSMA-derived epitopes when priming and boosting is performed with sPSMA. In contrast, tPSMA-transfected DCs prime T cells toward several PSMA-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance.

Statement from the Consensus Conference on Anti-D Prophylaxis

This article is also accessible online at: http://BioMedNet.com/karger Dr. S. Urbaniak Royal College of Physicians of Edinburgh 9 Queen Street Edinburgh EH2 1JQ UK At a conference convened by the Royal College of Physicians of Edinburgh and the Royal College of Obstetricians and Gynaecologists, a consensus panel considered specific issues relating to anti-D prophylaxis in the UK. This statement is based on presentations given at the meeting, published research and expert opinion. The panel reached the following conclusions:

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called “secreted” vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression. Monocyte-derived dendritic cells are transiently transfected with either sVac or one of two tVacs. The DCs are then used to activate CD25+-depleted or nondepleted autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs, peptide-pulsed DCs or monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tVacDCs and sVacDCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted towards one of the three antigen-derived epitopes when priming and boosting is performed with sVacDCs. In contrast, tVac-transfected DCs prime T cells towards all antigen-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance. While CD25+ cell depletion prior to priming with sVacDCs alleviates immunodominance, cotransfection of dendritic cells with GITR-L does so in some but not all cases.