David M. Kofler

In Vitro Selection of Viral Vectors with Modified Tropism: The Adeno-associated Virus Display

Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.

Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking

ABSTRACT To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.

CD28 Costimulation Overcomes Transforming Growth Factor-β–Mediated Repression of Proliferation of Redirected Human CD4+ and CD8+ T Cells in an Antitumor Cell Attack

The T-cell-mediated antitumor immune response is frequently repressed in the tumor environment by an immunologic barrier, the predominant mediators of which are thought to be interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). We explored the effect of these cytokines on the individual T-cell effector functions on antigen engagement during an antitumor cell attack. Isolated CD4+ and CD8+ T cells were antigen-specifically redirected toward carcinoembryonic antigen (CEA)-positive tumor cells by expression of a recombinant T-cell receptor (immunoreceptor), which triggers T-cell activation via CD3zeta on binding to CEA. Immunoreceptor-activated T cells secrete IFN-gamma, proliferate, and lyse CEA+ but not CEA- tumor cells. Whereas IL-10 has no direct effect on immunoreceptor-triggered effector functions, TGF-beta represses proliferation of both CD4+ and CD8+ T cells but neither IFN-gamma secretion nor specific cytolytic activities. CD28 costimulation, however, overcomes TGF-beta-mediated repression in T-cell proliferation. Consequently, T cells redirected by a combined CD28-CD3zeta signaling immunoreceptor are largely resistant to TGF-beta-mediated repression. This is reflected in vivo by a more pronounced antitumor activity of T cells against TGF-beta-secreting tumors when redirected by a costimulatory CD28-CD3zeta than by a CD3zeta signaling immunoreceptor.

The potential of gene transfer into primary B-CLL cells using recombinant virus vectors.

Despite recent advances, chronic lymphocytic leukemia (CLL) as the most common leukemia remains a largely incurable disease. Modern treatment options include novel drugs like purine analogues, monoclonal antibodies and transplantation strategies. Moreover, gene transfer of immunostimulatory molecules is another, but still experimental approach that can be used to potentiate immune responses against leukemic cells. CD40 ligand (CD40L) was shown to be a promising molecule for immunotherapy of B-CLL playing a critical role in immune activation. However, CLL B cells are resistant to transduction with most currently available vector systems. Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy in this area. Using replication defective adenovirus encoding CD40L (Ad-CD40L), immunologic and clinical responses were seen in CLL patients after infusion of autologous Ad-CD40L-CLL cells in a recent phase I trial. Due to the immunogenic nature of adenovirus vectors, alternative vector systems are currently explored. Recombinant adeno-associated virus (rAAV) was shown to enable efficient transduction of primary B-CLL cells. By use of a library of AAV clones with randomly modified capsids, receptor-targeting mutants with a tropism for CLL cells can be selected. Furthermore, helper-virus free Epstein-Barr virus (EBV)-based gene transfer vectors hold promise for development of CLL-targeted vaccines after remaining safety issues will be resolved. Herpes simplex virus (HSV)-based vectors, especially HSV amplicons, have favorable features for B-CLL gene transfer including high transduction efficiency, ability to infect postmitotic cells and a large packaging capacity. The challenge for the future will be to transfer these alternative vector systems into clinic and allow the detection of a CLL-specific immune response by use of defined tumor antigens. This will make it possible to establish the potential clinical role of gene therapy for CLL patients.

Current status of Immunotherapy in B Cell Malignancies

Conventional treatment of hematologic malignancies mainly consists of chemotherapeutic agents or a combination of both, chemotherapy and monoclonal antibodies. Despite recent advances, chemotherapeutic treatments often remain unsatisfying due to severe side effects and incomplete long-term remission. Therefore the evaluation of novel therapeutic options is of great interest. B cell malignancies, in particularly follicular lymphomas, chronic lymphocytic leukemia and multiple myeloma, represent the most immune-responsive types of all human cancer. Several immunotherapeutic strategies are presently employed to combat these B-cell malignancies. Active immunotherapies include vaccination strategies with dendritic cells (DCs) and genetically-modified tumor cell preparations as well as DNA and protein vaccination. Most of these vaccines target the tumor-specific immunoglobulin idiotype and have already demonstrated some anti-lymphoma activity in early phase clinical trials while their definitive impact is evaluated in ongoing phase III randomized trials. In contrast to these active immunizations, T cells transduced with chimeric antigen receptors and donor leukocyte infusions (DLI) represent adoptive (passive) immunotherapies. Recent advances of gene transduction technologies enabled improvement of immunotherapeutic strategies based on genetic modification of malignant cells or adoptive T cells. Current early phase clinical trials are investigating the potential of these innovative approaches. At the moment it remains unclear if the novel immunotherapeutic strategies will be able to play a similar role in the treatment of B cell malignancies than the already established antibody-based immunotherapy.

High level of transgene expression in primary chronic lymphocytic leukemia cells using helper-virus-free recombinant Epstein-Barr virus vectors.

OBJECTIVE Epstein-Barr virus (EBV)-based vectors have favorable features for gene transfer, including a high transduction efficiency especially for B cells, large packaging capacity up to 150 kb pairs, and ability to infect postmitotic cells. Recombinant EBV was explored for transduction of primary human B-cell chronic lymphocytic leukemia (CLL) cells. MATERIAL AND METHODS EBV vectors deleted for all oncogenic sequences and encoding terminal repeats (TR) essential for encapsidation, the lytic origin of replication (oriLyt) for DNA amplification, and the enhanced green fluorescent protein (EGFP) were packaged using an optimized, helper-virus-free method. Infectious EBV virions encoding EGFP (EBV/EGFP) with an infectious titer up to 2 x 10(6) per milliliter were generated. Primary leukemic cells from 14 patients with CLL were successfully transduced with EBV/EGFP at a very low multiplicity of infection (< 1). RESULTS Transgene expression was detected in up to 85% of cells 48 hours after infection. Transduction was specifically mediated by EBV vectors because gene transfer was inhibited by an antibody (72A1) directed against the viral envelope glycoprotein gp350/220. Furthermore, transduction of CLL cells with packaged EBV vectors coding for EGFP but deleted for TR sequences (TR-) did not result in EGFP expression compared to TR+ vector constructs (p = 0.009). CONCLUSION Helper-virus-free EBV-based gene transfer vectors hold promise for development of genetic therapies for CLL patients.

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.