Denise R. Shaw

Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

ABSTRACT Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

Adeno-Associated Virus 2-Mediated Antiangiogenic Cancer Gene Therapy: Long-Term Efficacy of a Vector Encoding Angiostatin and Endostatin over Vectors Encoding a Single Factor

Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy.

Expression of sperm protein 17 (Sp17) in ovarian cancer

Sperm protein 17 (Sp17) is an antigenic protein highly expressed in spermatozoa. Sp17 expression was demonstrated recently in multiple myeloma, suggesting that it may be a novel cancer‐testis antigen. Expression of Sp17 mRNA and protein was examined in human ovarian tumors. Sp17 mRNA was evaluated by reverse transcription‐polymerase chain reaction (RT‐PCR) and Northern blot analysis of RNA derived from epithelial ovarian tumors and normal tissues. RT‐PCR analysis detected Sp17 transcripts in 15 of 18 (83%) primary ovarian tumors. The transcript was not detected in RNA derived from normal uterus or cervix, whereas weak expression was noted in some normal ovarian tissue samples. Northern blot analysis showed no detectable Sp17 mRNA expression in normal tissues, including normal ovary, but showed Sp17 expression in 17 of 25 ovarian tumors (68%). To evaluate protein expression, mouse monoclonal antibodies were produced against recombinant Sp17 protein and used in Western blot and immunohistochemical analyses of normal reproductive tissue and primary ovarian tumor samples. Sp17 protein was detected by Western blot analysis in normal spermatozoa and in 8 of 19 ovarian tumor samples. Immunohistochemical studies showed Sp17 expression in spermatozoa, ciliated cells of the female reproductive tract, and most ovarian tumors evaluated. Tumors showed a predominately nuclear localization of Sp17 expression, with some cytoplasmic staining. These results demonstrate that Sp17, a protein with restricted expression in somatic tissues, is expressed in ovarian tumors. Because Sp17 is immunogenic, it may represent a novel target for immunotherapeutic interventions for ovarian cancer patients.

Characterization of human mesothelin transcripts in ovarian and pancreatic cancer

BackgroundMesothelin is an attractive target for cancer immunotherapy due to its restricted expression in normal tissues and high level expression in several tumor types including ovarian and pancreatic adenocarcinomas. Three mesothelin transcript variants have been reported, but their relative expression in normal tissues and tumors has been poorly characterized. The goal of the present study was to clarify which mesothelin transcript variants are commonly expressed in human tumors.MethodsHuman genomic and EST nucleotide sequences in the public databases were used to evaluate sequences reported for the three mesothelin transcript variants in silico. Subsequently, RNA samples from normal ovary, ovarian and pancreatic carcinoma cell lines, and primary ovarian tumors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing to directly identify expressed transcripts.ResultsIn silico comparisons of genomic DNA sequences with available EST sequences supported expression of mesothelin transcript variants 1 and 3, but there were no sequence matches for transcript variant 2. Newly-derived nucleotide sequences of RT-PCR products from tissues and cell lines corresponded to mesothelin transcript variant 1. Mesothelin transcript variant 2 was not detected. Transcript variant 3 was observed as a small percentage of total mesothelin amplification products from all studied cell lines and tissues. Fractionation of nuclear and cytoplasmic RNA indicated that variant 3 was present primarily in the nuclear fraction. Thus, mesothelin transcript variant 3 may represent incompletely processed hnRNA.ConclusionMesothelin transcript variant 1 represents the predominant mature mRNA species expressed by both normal and tumor cells. This conclusion should be important for future development of cancer immunotherapies, diagnostic tests, and gene microarray studies targeting mesothelin.

Evaluation of a selectively oncolytic adenovirus for local and systemic treatment of cervical cancer.

Treatment options for disseminated cervical cancer remain inadequate. Here, we investigated a strategy featuring Ad5‐Δ24RGD, an oncolytic adenovirus replication‐competent selectively in cells defective in the Rb‐p16 pathway, such as most cervical cancer cells. The viral fiber contains an αvβ3 and αvβ5 integrin‐binding RGD‐4C motif, allowing coxsackie‐adenovirus receptor‐independent infection. These integrins have been reported to be frequently upregulated in cervical cancer. Oncolysis of cervical cancer cells was similar to a wild‐type control in vitro. In an animal model of cervical cancer, the therapeutic efficacy of Ad5‐Δ24RGD could be demonstrated for both intratumoral and intravenous application routes. Biodistribution was determined following intravenous administration to mice. Further preclinical safety data were obtained by demonstrating lack of replication of the agent in human peripheral blood mononuclear cells. These results suggest that Ad5‐Δ24RGD could be useful for local or systemic treatment of cervical cancer in patients with disease resistant to currently available modalities.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

Antitumor activity of the intratumoral injection of fowlpox vectors expressing a triad of costimulatory molecules and granulocyte/macrophage colony stimulating factor in mesothelioma

Deficiency in costimulatory molecule expression has been implicated in the ability of tumors to escape immune effectors. The activity of the intratumoral administration of recombinant fowlpox vectors expressing a triad of costimulatory molecules (rF‐TRICOM) was evaluated in the asbestos‐induced AB12 and AC29 mouse models of mesothelioma. Mesothelioma cell infected with rF‐TRICOM expressed high levels of the costimulatory molecules. Prolongation of survival was observed in mice receiving rF‐TRICOM in AB12 and AC29 intraperitoneal models. Complete tumor regressions were observed in mice receiving intratumoral rF‐TRICOM in the AB12 subcutaneous tumor model. Tumor regressions were associated with the development of serum IgG reactivities to mesothelioma‐associated determinants and specific systemic cytolytic activity, and responding mice were capable of rejecting tumors upon re‐challenge. Antitumor activity was also observed in mice with established AB12 tumor vaccinated with irradiated rF‐TRICOM‐infected AB12 cells. The antitumor activity of intratumoral rF‐TRICOM was superior to that of the intratumoral injection of a fowlpox vector expressing granulocyte‐macrophage colony stimulating factor (rF‐GM‐CSF). AB12 and AC29 tumors were found to produce GM‐CSF and to have substantial macrophage infiltration. Production of GM‐CSF decreased in vivo in tumors injected with rF‐TRICOM. rF‐TRICOM and wild‐type fowlpox inhibited the growth of AB12 and AC29 cells in vitro; less inhibition was observed with rF‐GM‐CSF. These results indicate that the intratumoral injection of rF‐TRICOM has significant activity in mouse models of mesothelioma and can elicit a systemic antitumor immune response. The results also suggest potential limitations to the intratumoral administration of cytokines, such as GM‐CSF, in mesothelioma.

Mesothelin: A New Target for Immunotherapy

To the Editor: The Perspective by Hassan et al. [(1)][1] is an excellent synopsis of the substantial studies by these authors and others that have identified mesothelin as a new target for tumor immunotherapy and diagnosis. We would like to comment on the mesothelin variants diagrammed in Fig. 2 of

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.