Georg-Speyer-Haus
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Objective: Establishment of an immunocompetent mouse model representing the typical progressive stages observed in malignant human gliomas for the in vivo evaluation of novel target-specific regimens.
Methods: Isolated clones from tumours that arose spontaneously in GFAP-v-src transgenic mice were used to develop a transplantable brain tumour model in syngeneic B6C3F1 mice. STAT3 protein was knocked down by infection of tumour cells with replication-defective lentivirus encoding STAT3-siRNA. Apoptosis is designed to be induced by soluble recombinant TRAIL + chemical Bcl-2/Bcl-xL inhibitors.
Results: Striatal implantation of 105 mouse tumour cells resulted in the robust development of microscopically (2 – 3 mm) infiltrating malignant gliomas. Immunohistochemically, the gliomas displayed the astroglial marker GFAP and the oncogenic form of STAT3 (Tyr-705-phosphorylated) which is found in many malignancies including gliomas. Phosphorylated STAT3 was particularly prominent in the nucleus but was also found at the plasma membrane of peripherally infiltrating glioma cells. To evaluate the role of STAT3 in tumour progression, we stably expressed siRNA against STAT3 in several murine glioma cell lines. The effect of STAT3 depletion on proliferation, invasion and survival will be first assessed in vitro and subsequently after transplantation in vivo. Upstream and downstream components of the STAT3 signalling pathway as well as possible non-specific side effects of STAT3-siRNA expression after lentiviral infection will be examined, too.
Conclusions: Its high rate of engraftment, its similarity to the malignant glioma of origin, and its rapid locally invasive growth should make this murine model useful in testing novel therapies for malignant gliomas.
Survivin functions as an apoptosis inhibitor and a regulator of cell division during development and tumorigenesis. Since survivin is a highly relevant target for tumor therapy, we investigated whether interference with it’s dynamic cellular localization represents a novel strategy to inhibit survivin’s cancer promoting functions. We confirmed survivin overexpression in head and neck as well as in colorectal cancers and identified an evolutionary conserved Crm1-dependent nuclear export signal (NES) in survivin. Importantly, nuclear export was required for survivin mediated protection against chemo- and radiotherapy-induced apoptosis by securing efficient interference with cytoplasmic caspases. In dividing cells, the NES was required for tethering of survivin and of the survivin/Aurora-B kinase complex to the mitotic machinery, which was inevitable for proper cell division. The clinical relevance of our findings was supported by showing that preferential nuclear localization of survivin correlated with enhanced survival in a cohort of colorectal cancer patients. Targeting survivin’s nuclear export by the application of NES-specific antibodies promoted its nuclear accumulation and inhibited its cytoprotective function. We here show that nuclear export is essential for the tumor promoting activities of survivin and encourage the identification of chemical inhibitors to specifically interfere with survivin’s nuclear export as a novel class of anticancer therapeutics.
Following publication of the data presented by von Minckwitz and colleagues it has been brought to our attention that some patients should be scored differently. Stable disease was seen in three of the eighteen patients instead of two of the eighteen patients: one patient with transitional cell carcinoma treated at 4 µg/kg scFv(FRP5)-ETA per day, and two breast cancer patients treated at 4 and 12.5 µg/kg scFv(FRP5)-ETA per day. Disease progression occured in 9 of the eighteen patients evaluated (see corrected Table 2 overleaf). This does not affect the conclusions of our study. In addition we would like to correct the following errors: patient IDs for patients U01 and U02 in the original Table 2 were interchanged. In addition, patient N03 had a grade 3 elevation of gamma-glutamyl transferase, and not grade 2 (see corrected Table 2 overleaf).
Phage display selection of HIV specific conserved mimotopes with IgG from long-term non-progressors
(2005)
Poster presentation Background The aim of this study is to identify conserved epitopes of HIV-1 neutralizing antibodies in polyclonal plasma from LTNP to finally derive vaccine candidates. Materials and methods The presence of neutralizing antibodies in 9 LTNP sera was proved by in vitro neutralization assays. Phage displayed peptide libraries were screened with LTNP IgG. HIV-specific mimotopes were analyzed for homology to the gp120 structure by a software (3DEX) especially developed for this purpose. Mice were immunized with interesting phages and their sera were analyzed for neutralizing activities against HIV-1. Results After biopannings, between 19% and 75% HIV-specific phage clones were identified by ELISA. Mimotope sequences were identified and could be aligned by 3DEX to linear or conformational epitopes on gp120. A peptide specific immune response was detected in sera of immunized mice. The first mice sera analyzed showed neutralizing activities against HIV-1. Conclusion Mimotopes could be selected from LTNP sera that represent conformational epitopes on gp120. Those ones inducing neutralizing antibodies upon immunization potentially are suited to derive vaccine candidates.
The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with retroviral vectors we have generated genetically modified NK-92 cells expressing chimeric antigen receptors specific either for the tumor-associated ErbB2 (HER2/neu) antigen or the human Epithelial Cell Adhesion Molecule (Ep-CAM). Both antigens are overexpressed by many tumors of epithelial origin. The chimeric antigen receptors consist of either the ErbB2 specific scFv(FRP5) antibody fragment or the Ep-CAM specific scFv(MOC31), a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. Transduced NK-92-scFv(FRP5)-zeta or NK-92-scFv(MOC31)-zeta cells express high levels of the fusion proteins on the cell surface as determined by FACS analysis. In europium release assays no difference in cytotoxic activity of NK-92 and transduced NK-92 cells towards ErbB2 or Ep-CAM negative targets was found. However, even at low effector to target ratios transduced NK-92 cells specifically and efficiently lysed established ErbB2 or Ep-CAM expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. Similarly, ErbB2-positive primary breast cancer cells isolated from pleural effusions of patients with recurrent disease were selectively killed by NK-92-scFv(FRP5)-zeta. In an in vivo model in immunodeficient mice treatment with retargeted NK-92-scFv(FRP5)-zeta, but not parental NK-92 cells resulted in markedly delayed growth of ErbB2 transformed cancer cells. These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2 and Ep-CAM expressing malignancies. This therapeutic approach might be applicable for a large variety of different cancers where suitable cell surface antigens have been identified.
The receptor tyrosine kinase ErbB2 (HER2) is overexpressed in multiple human tumors of epithelial origin. High ErbB2 expression is functionally involved in tumorigenesis and correlates with poor clinical prognosis. For immunotherapy of ErbB2 expressing tumors, we developed a strategy to supply the tumor cells with costimulatory activity. A bispecific fusion protein was constructed (BIg5), containing the IgV-like domain of huCD86, the CH2/CH3 domain of huIgG1 and the ErbB2-specific single chain antibody fragment scFv(FRP5). A similar fusion protein lacking the CD86 domain (Ig5) was used as a control. Upon binding of BIg5 to ErbB2 on tumor cells, these cells display CD86 on their surface and thus can deliver costimulatory signals for T-cell activation. In addition, NK cells could be activated by CD86 binding to CD28. BIg5 is secreted by eukaryotic cells as a homodimer with increased stability compared to monomers and possibly enhanced costimulatory activity due to crosslinking of CD28 on effector cells. By FACS analysis, specific binding of the scFv(FRP5) domain to ErbB2 as well as CD86 IgV binding to CTLA-4 could be demonstrated. Together with anti-CD3 antibody, BIg5 stimulates proliferation of human CD2-purified lymphocytes in vitro. After binding to ErbB2 on murine Renca-lacZ/ErbB2 tumor cells, about 50% of initially bound BIg5 is still present on the cell surface after 4 hours. For delivery of chimeric fusion proteins in vivo, we used syngeneic, stably transfected HC11 mammary epithelial cells continuously secreting the proteins. Inoculation of these bystander cells close to subcutaneously growing Renca-lacZ/ErbB2 tumors should provide a long-lasting source to achieve high local concentrations of BIg5 at the tumor site. In vivo HC11-BIg5 cells proved to be non-tumorigenic and secreted BIg5 for several weeks, causing a strong anti-BIg5 antibody response. Treatment of established Renca-lacZ/ErbB2 or ErbB2-negative Renca-lacZ tumors by peritumoral inoculation of either HC11-BIg5 or HC11-Ig5 cells led to rejection of all Renca-lacZ/ErbB2, but none of the Renca-lacZ tumors. HC11neo control cells had no effect on tumor growth. Rejection of ErbB2+ tumors led to long-term protection also against subsequent challenge with intravenously injected ErbB2- tumor cells. Intraperitoneal injection of bystander cells secreting the fusion proteins did not lead to tumor regression suggesting that high local concentrations at the tumor site are necessary to target ErbB2 on tumor cells and to overcome elimination of BIg5 or Ig5 by neutralizing antibodies. The CD86 IgV domain of BIg5 did not play a major role in the observed antitumoral immune response suggesting NK-cell mediated ADCC as the initial effector mechanism followed by activation of tumor specific T cells. Targeting of ErbB2 on tumor cells with antibody fusion proteins that interact specifically with the host immune system could be an efficient and specific approach for therapy of solid ErbB2+ tumors.
Tumor-specific T lymphocytes can be regarded as a highly effective mechanism for tumor rejection. A substantial number of T-cell defined tumor antigens including mutated oncoproteins and differentiation antigens have been identified. However, while most spontaneous tumors appear to be antigenic, few are immunogenic. Activation of tumor-specific cytotoxic T cells (CTL) requires presentation of tumor antigens by professional antigen presenting cells (APCs) via MHC I molecules. Due to their crucial role in T-cell activation, APCs are being exploited for active cancer immunotherapy. Present experimental strategies include the incubation of dendritic cells with synthetic, tumor specific peptides to achieve uptake of tumor antigens and presentation in the context of MHC molecules. Alternatively, gene therapeutic approaches are aimed at the endogenous expression of tumor antigens in APCs upon transfer of suitable vector constructs. Our strategy for the presentation of tumor antigens by APCs is based on the intracellular delivery of tumor antigens as part of a fusion protein specifically targeted to APC cell surface receptors. We have constructed prototype molecules that contain a soluble fragment of CTLA-4 for cell binding via interaction with B7 molecules, genetically fused to a protein fragment derived from the tumor-associated antigen ErbB2. To improve uptake and direct the antigenic determinant preferentially to the MHC class I pathway, in one of these protein vaccines also the translocation domain of the bacterial Pseudomonas exotoxin A has been included. In the parental toxin this protein domain facilitates escape from the endosomal compartment to the cytosol upon receptor mediated endocytosis. Here we have investigated the in vitro cell binding activity of such reagents and their antitumoral activity in immunocompetent murine model systems. Specific binding to B7 molecules and uptake of bacterially expressed protein vaccines could be demonstrated. Ex vivo restimulation with an ErbB2-derived peptide of splenocytes from Balb/c mice injected with the fusion proteins resulted in enhanced IFN-gamma production by T cells. Protective and therapeutic effects of ErbB2 protein vaccines were also investigated. Vaccinated animals were protected against subsequent challenge with syngeneic ErbB2 expressing tumor cells. Likewise, s.c. injection of ErbB2 protein vaccines in the vicinity of established tumors resulted in tumor rejection and long lasting protection indicating that immunological memory was induced. Our results suggest that chimeric proteins combining a tumor antigen and specific recognition of APCs in a single molecule are suitable for targeted delivery of antigens to professional APCs and might become valuable tools for cancer immunotherapy.
Oral presentations Background: We selected peptide ligands for the HIV-1 packaging signal PSI by screening phage displayed peptide libraries. Peptide ligands were optimized by screening spot synthesis peptide membranes. The aim of this study is the functional characterization of these peptide ligands with respect to inhibition of HIV-1 replication. Methods: Phage displayed peptide libraries were screened with PSI-RNA structures. The Trp-rich peptide motifs were optimized for specific binding on spot synthesis peptide membranes. The best binding peptide was expressed intracellularly in fusion with RFP or linked to a protein transduction domain (PTD) for intracellular delivery. The effects on virion production were analyzed using pseudotyped lentiviral particles. Results: After positive and negative selection rounds, phages binding specifically to PSI-RNA were identified by ELISA. Peptide inserts contained conserved motifs of aromatic amino acids known to be implicated in binding of PSI-RNA by the natural Gag ligand. The filter assay identified HKWPWW as the best binding ligand for PSI-RNA, which is delivered into several cell lines by addition of a PTD. Compared to a control peptide, the HKWPWW peptide inhibited HIV-1 replication as deduced from reduced titers of culture supernatants. As HKWPWW also binds to the TAR-RNA like the natural nucleocapsid PSI-RNA ligand, the effect on Tat-TAR inhibition will also be analyzed. Currently T-cell lines are established which stably express HKWPWW as well as a control peptide, which will be infected with HIV-1 to monitor the ability of HKWPWW to inhibit wild type HIV-1 replication. Conclusion: The selection of a peptide ligand for PSI-RNA able to inhibit HIV-1 replication proves the suitability of the phage display technology for the selection of peptides binding to RNA-structures. This enables the indentification of peptides serving as leads to interfere with additional targets in the HIV-1 replication cycle.
Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture
(1999)
Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki-ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki-ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.