Phase-I clinical trial of IL-12 plasmid/lipopolymer complexes for the treatment of recurrent ovarian cancer.

A phase-I trial to assess the safety and tolerability of human interleukin-12 (IL-12) plasmid (phIL-12) formulated with a synthetic lipopolymer, polyethyleneglycol-polyethyleneimine-cholesterol (PPC), was conducted on women with chemotherapy-resistant recurrent ovarian cancer. A total of 13 patients were enrolled in four dose-escalating cohorts and treated with 0.6, 3, 12 or 24 mg m(-2) of the formulated plasmid once every week for 4 weeks. Administration of phIL-12/PPC was generally safe and well-tolerated. Common side effects included low-grade fever and abdominal pain. Stable disease and reduction in serum CA-125 levels were clinically observed in some patients. Measurable levels of IL-12 plasmid were detectable in PF samples collected throughout the course of phIL-12/PPC treatment. In comparison, serum samples either did not contain detectable amounts of plasmid DNA or contained <1% of the amount found in the corresponding PF samples. Treatment-related increases in IFN-gamma levels were observed in PF but not in serum. These data demonstrate that IL-12 gene delivery with a synthetic delivery system is feasible for ovarian cancer patients.

Early resynchronization of non-pregnant beef cows based in corpus luteum blood flow evaluation 21 days after Timed-AI.

The study aimed to verify whether a hormone protocol started at Day 13 (D13) after Timed Artificial Insemination (TAI) influences the conception rate. Nelore cows (primiparous and multiparous) from two commercial beef farms (n = 1,431) were first TAI (D0). Timed AI was performed in lots (TAI Lots) ranging from 187 to 346 cows. On D13, regarding the TAI lot, cows were assigned for either receiving (Resynch group, n = 1,002) or not (Control group, a subset of approximately 30%, n = 429) another hormone protocol for resynchronization. The same hormone protocol was used for the first TAI and for the resynchronization, except for 1 mg instead of 2 mg of estradiol benzoate (EB) at the begging of the protocol. Eight days later (D21), the Resynch group was checked for corpus luteum blood flow by color Doppler ultrasonography, and in those detected as non-pregnant, the protocol was completed and a 2nd TAI was performed at D23. Pregnancy diagnosis was later (D30) performed by B-mode ultrasonography in the control group and confirmed in the presumptive pregnant cows from the 1st TAI of the Resynch group. The remaining cows were checked for pregnancy 30 days after the 2nd TAI (experimental Day 53). The statistical model to explain conception rate considered the effects of Group (Control or Resynch), Farm, Parity (primiparous or multiparous), Sire, Technician (who perform AI), TAI Lot and pertinent interactions (Group*Parity, Group*Farm and Group*TAI Lot). The statistical analyses of the model were performed using the Proc Glimmix (SAS virtual University Edition). The conception rate for the 1st TAI was similar (P > 0.4) between Control (50.3%, 216/429) and Resynch group (52.6%, 527/1002). The positive predictive diagnostic on D21 showed high relation with PD30 (90.7%, 527/581). In Resynch group, non-pregnant cows (n = 421, 1002 minus 581) were re-inseminated. The conception rate of the 2nd TAI (42.8%, 180/421) was affected (P < 0.002) by side effects of the Farm (48.5 vs. 33.1%) and Parity (51.2 vs. 40.3%, for multiparous vs. primiparous, p < 0.001). Nevertheless, after the 2 TAIs of the Resynch group, the cumulative conception rate was 70.5% (707/1002). In conclusion, the early resynchronization of cows with a low (1 mg) EB dose and progesterone device at D13 after TAI can be used as a strategy to reduce conception interval in beef cattle, and thus to increase the number of pregnant cows from artificial insemination after the breeding season.

Cluster intradermal DNA vaccination rapidly induces E7-specific CD8+ T-cell immune responses leading to therapeutic antitumor effects.

Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD+ T-cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal time frame. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T-cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T-cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T-regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor-infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation.

A multi-institutional evaluation of factors predictive of toxicity and efficacy of bevacizumab for recurrent ovarian cancer.

While bevacizumab has shown activity in recurrent ovarian cancer, a higher than expected incidence of bowel perforations has been reported in recent trials. We sought to determine factors associated with toxicity and tumor response in patients with relapsed ovarian cancer treated with bevacizumab. A retrospective review of patients with recurrent ovarian cancer treated with bevacizumab was undertaken. Response was determined radiographically and through CA125 measurements. Statistical analysis to determine factors associated with toxicity and response was performed. Sixty-two eligible patients were identified. The cohort had received a median of 5 prior chemotherapy regimens. Single-agent bevacizumab was administered to 12 (19%), while 50 (81%) received the drug in combination with a cytotoxic agent. Grade 3-5 toxicities occurred in 15 (24%) patients, including grade 3-4 hypertension in 4 (7%), gastrointestinal perforations in 7%, and chylous ascites in 5%. Development of chylous ascites and gastrointestinal perforations appeared to correlate with tumor response. The overall response rate was 36% (4 complete response, 17 partial response), with stable disease in 40%. A higher objective response rate was seen in the bevacizumab combination group compared to single-agent treatment (43% vs 10%) (P = 0.07). However, 29 grade 3-5 toxic episodes were seen in the combination group vs only 1 in the single-agent bevacizumab cohort (P = 0.071). We conclude that bevacizumab demonstrates promising activity in recurrent ovarian cancer. The addition of a cytotoxic agent to bevacizumab improved response rates at the cost of increased toxicity. Gastrointestinal perforations occurred in 7%. The perforations occurred in heavily pretreated patients who were responding to therapy.

Targeted tumor killing via an intracellular antibody against erbB-2.

Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis.

Intracellular expression of a single-chain antibody directed against human papillomavirus type 16 E7 oncoprotein achieves targeted antineoplastic effects.

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. Anti-HPV16 E7 intracellular single-chain antibodies (scFvs) were constructed to down-regulate HPV16 E7 oncoprotein in HPV DNA-containing cell lines. In these studies, we transfected anti-E7 scFvs into the HPV16-positive human cervical carcinoma cell lines CaSki and SiHa and tested them for their ability to inhibit cell proliferation and alter the level of HPV16 E7 oncoprotein. Our results showed that anti-HPV16 E7 scFvs inhibited cell proliferation by >85% in CaSki cells and by 95% in SiHa cells. E7 oncoprotein was down-regulated by anti-HPV16 E7 scFv, and its expression was inversely related to the amount of scFv transfected. However, there were no effects of transfecting scFvs alone in HPV-negative cell lines. These results imply that anti-HPV16 E7 scFvs only have specific anti-HPV16 E7 effects on cell proliferation and on the synthesis of virally encoded proteins in HPV-positive cell lines. Thus, transfection of HPV16 E7-positive tumors with antigen-specific scFvs may be a viable strategy for cervical cancer gene therapy.

Phase III randomized study of cisplatin versus paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage III or IV ovarian cancer: a gynecologic oncology group study.

PURPOSE: To assess progression-free survival (PFS) and overall survival (OS) in patients with suboptimally debulked epithelial ovarian cancer receiving cisplatin (100 mg/m(2)) or 24-hour infusion paclitaxel (200 mg/m(2)) or the combination of paclitaxel (135 mg/m(2)) followed by cisplatin (75 mg/m(2)). PATIENTS AND METHODS: After stratification for disease measurability, patients were randomized to receive six cycles of one of the treatments every 3 weeks. If measurable, complete response (CR) or partial response (PR) was determined. RESULTS: Six hundred fourteen of 648 patients who entered onto the trial were eligible. Monotherapies were discontinued more frequently (cisplatin because of toxicity or patient refusal [17%], and paclitaxel because of progression [20%]) compared with the combination therapy (7% and 6%, respectively). Neutropenia, fever, and alopecia were more severe with paclitaxel-containing regimens; whereas anemia, thrombocytopenia, neurotoxicity, nephrotoxicity, and gastrointestinal toxicity were more severe with cisplatin-containing regimens. The CR/PR rates on paclitaxel monotherapy were significantly lower compared with the cisplatin regimens (42% v 67%, respectively; P <.001). The relative hazard (RH) of first progression or death was significantly greater among those randomized to paclitaxel (RH = 1.41; 95% confidence interval [CI], 1.15 to 1.73; P <.001) when compared with cisplatin; however, RH did not differ significantly between the two cisplatin regimens (RH = 1.06; 95% CI, 0.895 to 1.30). Relative to cisplatin, the death rate on paclitaxel was 15% greater (RH = 1.15; 95% CI, 0. 929 to 1.42), and the death rate on the combination treatment was 1% less (RH = 0.99; 95% CI, 0.795 to 1.23). These differences among treatment groups were not statistically significant (P =.31). CONCLUSION: Cisplatin alone or in combination yielded superior response rates and PFS relative to paclitaxel. However, OS was similar in all three arms, and the combination therapy had a better toxicity profile. Therefore, the combination of cisplatin and paclitaxel remains the preferred initial treatment option.

Paclitaxel, an active agent in nonsquamous carcinomas of the uterine cervix: a Gynecologic Oncology Group Study.

PURPOSE: A phase II trial of paclitaxel was initiated in advanced nonsquamous carcinoma of the cervix to determine its activity in patients who had failed standard chemotherapy. PATIENTS AND METHODS: Eligible patients had at least one measurable lesion. The starting dose of paclitaxel was 170 mg/m(2) (135 mg/m(2) for patients with prior pelvic radiation) given as a 24-hour continuous intravenous infusion with courses repeated every 3 weeks. Dose escalation to 200 mg/m(2) and de-escalation to 110 mg/m(2) were allowed based on adverse effects. RESULTS: In this trial, 42 assessable patients were initially entered onto the study, and 13 responses were seen; four patients had a complete response, and nine patients had a partial response. The overall response rate was 31%. The primary and dose-limiting toxicity was neutropenia. CONCLUSION: The response rate to paclitaxel exceeds the rates reported using other single agents in nonsquamous carcinoma of the cervix.

Oral medroxyprogesterone acetate in the treatment of advanced or recurrent endometrial carcinoma: a dose-response study by the Gynecologic Oncology Group.

PURPOSE: Progestins have definite activity against advanced or recurrent endometrial carcinoma. Both parenteral and oral progestins yield similar serum levels and response rates, which range from 18% to 34%. The one major study that used oral medroxyprogesterone acetate (MPA) noted a response rate at the lower end of the range (18%) and much poorer progression-free and overall survival times (4 and 10.5 months, respectively) than previously reported. The present study sought to confirm this earlier study of oral MPA, to assess the importance of prognostic factors such as histologic grade and receptor levels, and to determine whether a higher dose of MPA would yield a higher response rate. PATIENTS AND METHODS: Two hundred ninety-nine eligible women with advanced or recurrent endometrial carcinoma were randomized to receive oral MPA either 200 mg/d or 1, 000 mg/d until unacceptable toxicity intervened or their disease progressed. RESULTS: Among 145 patients receiving the low-dose regimen, there were 25 complete (17%) and 11 partial (8%) responses for an overall response rate of 25%. The 154 patients receiving the high-dose regimen experienced 14 (9%) complete and 10 (6%) partial responses for an overall response rate of 15%. Median durations of progression-free survival were 3.2 months and 2.5 months for the low-dose and high-dose regimens, respectively. Median survival durations were 11.1 months and 7.0 months, respectively. The adjusted relative odds of responding to the high-dose regimen compared with the low-dose regimen was 0.61 (90% confidence interval, 0.36 to 1.04). Prognostic factors having a significant impact on the probability of response included initial performance status, age, histologic grade, and progesterone receptor concentration. Compliance with oral therapy was documented with serum levels 1 month after starting therapy, when possible. MPA levels were commensurate with the assigned dose and schedule. CONCLUSION: Oral MPA is active against endometrial carcinoma. Response to progestin therapy is more frequent among patients with a well-differentiated histology and positive progesterone receptor status. This study provides no evidence to support the use of MPA 1,000 mg/d orally instead of MPA 200 mg/d orally. In fact, the trends suggest the opposite. The use of oral MPA 200 mg/d is a reasonable initial approach to the treatment of advanced or recurrent endometrial carcinoma, particularly those lesions that are well-differentiated and/or progesterone receptor-positive (> 50 fmol/mg cytosol protein). Patients with poorly differentiated and/or progesterone receptor levels less than 50 fmol/mg cytosol protein had only an 8% to 9% response rate.

Phase I trial and pharmacologic trial of sequences of paclitaxel and topotecan in previously treated ovarian epithelial malignancies: a Gynecologic Oncology Group study.

PURPOSE: A phase I and pharmacologic study to evaluate the feasibility of administering paclitaxel (PTX) in combination with topotecan (TPT) without and with granulocyte colony-stimulating factor (G-CSF) in women with recurrent or refractory ovarian cancer. PATIENTS AND METHODS: TPT was administered as a 30-minute infusion daily for 5 days and PTX was given as a 24-hour infusion (PTX-24) either before TPT on day 1 or after TPT on day 5. Each patient received both schedules on an alternating basis every 3 weeks. Sequential dose escalation of TPT or PTX-24 without and with G-CSF resulted in five dosage permutations of TPT/PTX (mg/ m2): 0.75/135 without G-CSF and 0.75/135, 1.25/135, 1.50/135, and 1.25/170 with G-CSF. RESULTS: Twenty-two patients received 109 courses of therapy. Dose-limiting myelosuppression consistently occurred at the first TPT/PTX-24 dose level (0.75/135 mg/m2) in the absence of G-CSF support. Although the addition of G-CSF resulted in reduced rates of complicated neutropenia, the incidences of dose-limiting neutropenia and thrombocytopenia were unacceptably high after the doses of either TPT or PTX-24 were increased. Paired analysis showed similar hematologic toxicities between the two sequences of drug administration. The pharmacologic behavior of both TPT and PTX-24 was not altered by drug sequencing. Major antitumor responses occurred in 40% of patients with measurable and assessable disease, including 45% and 9% of patients with potentially cisplatin-sensitive and -resistant tumors, respectively. CONCLUSION: The recommended doses of TPT on a daily times-five schedule combined with PTX-24 in these patients were 0.75 mg/m2/d and 135 mg/m2, respectively, with G-CSF support. Although this dose of PTX has significant single-agent activity in ovarian cancer, the dose of TPT is much lower than the TPT dose at which single-agent activity has been observed. Due to the inability to administer near relevant single-agent doses of both drugs in combination, as well as the requirement for G-CSF support, further evaluations of this regimen in women with refractory or recurrent ovarian cancer are necessary before it can be recommended for previously treated patients in this setting.

Novel gene therapy strategy to accomplish growth factor modulation induces enhanced tumor cell chemosensitivity.

erbB-2 is a cell surface transmembrane glycoprotein which, when overexpressed, has been shown to be relevant to intrinsic tumor cell chemoresistance. Thus, strategies to down-regulate cell surface erbB-2 have resulted in enhanced tumor cell chemosensitivity. We have recently reported a gene therapy strategy to down-modulate erbB-2 expression using a plasmid construct encoding an intracellular single chain antibody. Therefore, we now demonstrate enhanced chemosensitivity to cis-diamminedichloroplatinum in erbB-2 overexpressing tumor cells and a model system of stable clones using an intracellular single chain antibody. These findings are consistent with the hypothesis that erbB-2 plays a role in tumor cell chemoresistance. In addition, these findings represent a novel gene therapy approach to overcome erbB-2-mediated tumor cell chemoresistance.

Selectivity of TAG-72-targeted adenovirus gene transfer to primary ovarian carcinoma cells versus autologous mesothelial cells in vitro.

Efficient gene transfer by recombinant adenovirus (Ad) vectors depends on expression of CAR and alpha(v) integrin on target cells. Because Ad may also infect nearby nontarget cells expressing these receptors, such as peritoneal mesothelial cells after i.p. injection, we hypothesized that targeting Ad gene delivery to a receptor overexpressed on most ovarian carcinoma cells, such as TAG-72, would enhance the selectivity of Ad gene transfer when used in this context. A monoclonal antibody that has been investigated clinically for immunotherapy and immunodetection of ovarian carcinomas, namely CC49, was used to construct a bispecific conjugate with the Fab fragment of a neutralizing anti-knob mAb to target Ad binding via TAG-72. This conjugate facilitated TAG-72-specific, CAR-independent Ad reporter gene transfer to both ovarian cancer cell lines and primary ovarian cancer cells cultured from malignant ascites fluid. Fab-CC49 was very selective for tumor cells, augmenting Ad gene transfer to primary ovarian cancer cells 2- to 28-fold relative to untargeted Ad, while also decreasing gene transfer to autologous cultured mesothelial cells 4- to 9-fold. These data suggest that targeting Ad via TAG-72 may improve the selectivity of Ad gene transfer for ovarian tumors 8- to 252-fold on i.p. vector injection. These results also define the requirements for a candidate target receptor in the rational design of a targeted Ad vector for ultimate clinical utility, one that selectively infects tumor cells and spares normal cells on i.p. injection. Such a vector may increase gene transfer and decrease the toxicity of Ad vectors, which would improve the therapeutic index of cytotoxic gene therapy for ovarian cancer in clinical trials.

Genetically modified CD34+ cells exert a cytotoxic bystander effect on human endothelial and cancer cells.

We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34+ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34+ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34+ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34+ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34+ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34+ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34+ cells and total cell killing. For example, when 50% of CD34+ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80% of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34+ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.

Endothelial cell vehicles for delivery of cytotoxic genes as a gene therapy approach for carcinoma of the ovary.

Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. After demonstrating that HUVECs could be transduced with the reporter gene LacZ encoded by an adenoviral vector, appropriate cell killing of the AdCMVHSV-TK-transduced HUVECs was exhibited after treatment with 20 microM ganciclovir. Mixing experiments were then performed to determine whether the transduced HUVECs would demonstrate a bystander effect with the ovarian cancer cell lines. When 50% AdCMVHSV-TK-transduced HUVECs were mixed with untransduced ovarian cancer cells, > 70% of all cells were killed. Finally, s.c. and i.p. injections of herpes simplex-thymidine kinase-expressing HUVECs and SKOV3ip1 tumor cells were performed to evaluate the effects of HUVECs in in vivo models. These studies showed a decrease in tumor growth s.c. as well as a statistically significant survival prolongation (P < 0.05) in the i.p. model. These findings suggest that endothelial cells may be used as a vehicle for the delivery of cytotoxicity (bystander effect) in molecular chemotherapy.

Adenoviral-mediated delivery of the herpes simplex virus thymidine kinase gene selectively sensitizes human ovarian carcinoma cells to ganciclovir.

One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.

Interleukin-6 modulated conditionally replicative adenovirus as an antitumor/cytotoxic agent for cancer therapy.

In this study, we report that an interleukin-6 (IL-6)-inducible E1A-substituting activity can be exploited for the production of infectious adenoviral particles during infection with the E1A-deleted adenovirus (Ad) Ad5dl312. The basal level of complementation can be increased by 1.5 log by induction of the HepG2 cells with recombinant human IL-6. Additionally, the IL-6-inducible E1A-substituting activity can complement E1A deletion in other cancer cell lines to render them Ad producer cells on induction with recombinant human IL-6, although the efficiency of complementation varies between cell lines. Ad5dl312 can replicate in, produce cytotoxic effect, and kill human tumor cells without addition of exogenous IL-6 in the context of tumor cells possessing an IL-6 autocrine arc, such as ovarian tumor cells. In contrast, normal human mesothelial cells isolated from normal human peritoneum lining do not support replication of Ad5dl312, even in the presence of exogenous IL-6. These results suggest that Ad5dl312 could be used as a cytotoxic agent to selectively kill tumor cells responsive to or possessing an IL-6 autocrine arc.

Basic fibroblast growth factor enhancement of adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene results in augmented therapeutic benefit in a murine model of ovarian cancer.

A number of preclinical and human clinical gene therapy trials using adenoviral vectors have shown that the number of viral particles necessary to give adequate levels of gene transfer can be associated with significant vector-related toxicity. In an effort to reduce the number of adenoviral particles required for a given level of gene transfer, we sought to redirect adenoviral infection via a receptor that is highly expressed on the target cells. By using basic fibroblast growth factor (FGF2) as the targeting ligand, adenovirus-mediated gene transfer to the human ovarian cancer cell line SKOV3.ip1 was significantly enhanced, permitting the transduction of a greater number of target cells to be achieved by a given dose of virus. In a murine model of human ovarian carcinoma, an FGF2-redirected adenoviral vector carrying the gene for herpes simplex virus thymidine kinase (AdCMVHSV-TK) was shown to result in a significant prolongation of survival compared with the same number of particles of unmodified AdCMVHSV-TK. In addition, equivalent survival rates were achieved with a 10-fold lower dose of the FGF2-redirected AdCMVHSV-TK compared with the unmodified vector. To our knowledge, this is the first report demonstrating that strategies to enhance the efficiency of in vivo transduction of adenoviral vectors will be of clinical utility.

A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial.

The purpose of this Phase I study was to determine the feasibility of using an anti-erbB-2-encoding adenovirus (Ad21) to treat erbB-2-overexpressing ovarian cancer. Recurrent ovarian cancer patients were treated i.p. with Ad21 in dosages ranging from 1 x 10(9) to 1 x 10(11) pfu. Patients were monitored after treatment for evidence of clinical toxicity and efficacy. Peritoneal aspirates and serum samples were obtained to assess for evidence of gene transfer/expression, for generation of wild-type vector, and antiadenoviral humoral response. Fifteen patients were treated per study specifications. Treatment-specific grade 1/2 fever was experienced by 9 of 15 (60%) patients. Other transient grade 1/2 constitutional, pain, and gastrointestinal symptoms were also experienced. No dose-limiting vector-related toxicity was experienced. Of 13 patients evaluable for response, 5 (38%) had stable disease and 8 (61%) had evidence of progressive disease. One patient with nonmeasurable disease normalized her CA125 at the 8-week evaluation, and one patient with nonmeasurable disease remained without clinical evidence of disease for 6 months after treatment. PCR analysis of peritoneal aspirates demonstrated the presence of Ad21 in 84.6%, 84.6%, and 61.6% of evaluable specimens at days 2, 14, and 56 after treatment, respectively. No wild-type virus was detected. Reverse transcription-PCR analysis demonstrated expression of the anti-erbB-2 sFv-encoding gene in 10 of 14 evaluable patients at day 2. Five of six evaluable patients had an increase in antiadenovirus antibody titer. This study suggests that adenoviral-mediated gene therapy using an anti-erbB-2-directed intrabody is feasible in the context of human ovarian cancer.

Adenovirus-mediated soluble FLT-1 gene therapy for ovarian carcinoma.

PURPOSE: We hypothesized that adenovirus-mediated soluble fms-like tyrosine kinase receptor (sFLT-1) gene therapy can inhibit the ovarian tumor growth and increase survival of mice in the context of ovarian carcinoma. EXPERIMENTAL DESIGN: We constructed an infectivity-enhanced recombinant adenovirus (AdRGDGFPsFLT-1) expressing soluble FLT-1 and green fluorescent protein (GFP). An adenovirus AdRGDGFP expressing GFP alone was used as control. The functional validation of adenovirus-mediated sFLT-1 was determined by an in vitro human umbilical vein endothelial cell proliferation inhibition assay. To evaluate the therapeutic potential of adenovirus-expressed sFLT-1 to inhibit the growth of ovarian tumors and to increase the survival duration of mice with ovarian tumors, two tumor models were used. First, SKOV3.ip1 ovarian carcinoma cells were infected ex vivo with either AdRGDGFPsFLT-1 or AdRGDGFP or uninfected and then inoculated s.c. into BALB/c nude mice, and tumor growth was monitored. Second, SKOV3.ip1 cells were inoculated i.p. into CB17 SCID mice and then treated with two doses of either AdRGDGFPsFLT-1 or AdRGDGFP or with PBS on days 1 and 14 after inoculation of cells, and the survival duration was monitored. RESULTS: Treatment with adenovirus-expressed sFLT-1 significantly inhibited the proliferation of human umbilical vein endothelial cells. The s.c. tumor nodules in mice derived from cells infected with AdRGDGFPsFLT-1 were significantly smaller than those infected with either AdRGDGFP or uninfected. In addition, i.p. administration of the AdRGDGFPsFLT-1 resulted in a significant increase in the survival times of mice compared with AdRGDGFP- or PBS-treated mice. CONCLUSIONS: Our results suggest that adenovirus-mediated sFLT-1 gene therapy can effectively inhibit ovarian tumor growth and increase survival in a murine model of ovarian carcinoma.

Strategies to accomplish targeted expression of transgenes in ovarian cancer for molecular therapeutic applications.

PURPOSE: The purpose of the study was to determine the capability of the midkine (MK) and cycooxygenase-2 (cox-2) gene promoter regions to function as tumor-specific promoters for use in targeted gene therapy of ovarian cancer. EXPERIMENTAL DESIGN: Established and primary ovarian cancer and mesothelial cells were transduced by adenoviral vectors containing a reporter or thymidine kinase gene expressed under the control of the MK, cox-2, or cytomegalovirus (CMV) promoters. SCID or C57BL/6 mice were injected i.p. with these same vectors. In vitro reporter gene expression and cellular cytotoxicity was determined using luciferase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Acute toxicity in vivo was assessed by histological evaluation of harvested tissues. RESULTS: Consistent activation of the MK and cox-2 promoters was noted in all of the ovarian cancer cell lines in addition to primary ovarian cancer cells. In contrast, reduced reporter activity was reported in mesothelial cells transduced with adenoviruses containing the test promoters, which was especially apparent for the cox-2 promoter. Additionally, the cox-2 promoter exhibited significantly lower reporter gene levels in liver and peritoneum than the control promoter in in vivo experiments. Tumor-cell killing induced by Adcox-2 MTK was comparable to that observed with AdCMVTK. However, a clear differential toxicity pattern was observed in favor of animals treated with Adcox-2 MTK when compared with controls. CONCLUSIONS: These data clearly demonstrate that the transcriptional control afforded by the cox-2 promoter is tumor-specific and is able to mitigate associated toxicity in normal tissue while maintaining therapeutic efficacy in the context of an ovarian cancer molecular chemotherapeutic approach.