Understanding and addressing barriers to successful adenovirus-based virotherapy for ovarian cancer.

Ovarian cancer is the leading cause of death among women with gynecological cancer, with an overall 5-year survival rate below 50% due to a lack of specific symptoms, late stage at time of diagnosis and a high rate of recurrence after standard therapy. A better understanding of heterogeneity, genetic mutations, biological behavior and immunosuppression in the tumor microenvironment have allowed the development of more effective therapies based on anti-angiogenic treatments, PARP and immune checkpoint inhibitors, adoptive cell therapies and oncolytic vectors. Oncolytic adenoviruses are commonly used platforms in cancer gene therapy that selectively replicate in tumor cells and at the same time are able to stimulate the immune system. In addition, they can be genetically modified to enhance their potency and overcome physical and immunological barriers. In this review we highlight the challenges of adenovirus-based oncolytic therapies targeting ovarian cancer and outline recent advances to improve their potential in combination with immunotherapies.

Targeting both tumour-associated CXCR2+ neutrophils and CCR2+ macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma.

OBJECTIVE: Chemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated CCR2+ macrophages (TAM) alone improves antitumour immunity in preclinical models. However, a compensatory influx of an alternative myeloid subset may result in a persistent immunosuppressive TME and promote therapeutic resistance. Here, we show CCR2 and CXCR2 combined blockade reduces total tumour-infiltrating myeloids, promoting a more robust antitumour immune response in PDAC compared with either strategy alone. METHODS: Blood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy. RESULTS: A systemic increase in CXCR2+ TAN correlates with poor prognosis in PDAC, and patients receiving CCR2i showed increased tumour-infiltrating CXCR2+ TAN following treatment. In an orthotopic PDAC model, CXCR2 blockade prevented neutrophil mobilisation from the circulation and augmented chemotherapeutic efficacy. However, depletion of either CXCR2+ TAN or CCR2+ TAM resulted in a compensatory response of the alternative myeloid subset, recapitulating human disease. This was overcome by combined CCR2i and CXCR2i, which augmented antitumour immunity and improved response to FOLFIRINOX chemotherapy. CONCLUSION: Dual targeting of CCR2+ TAM and CXCR2+ TAN improves antitumour immunity and chemotherapeutic response in PDAC compared with either strategy alone.

Mammaglobin-A is a target for breast cancer vaccination.

We recently completed a phase 1 clinical trial demonstrating the safety of a mammaglobin-A DNA vaccine in patients with metastatic breast cancer. We are currently enrolling patients with early stage breast cancer in a phase 1b clinical trial. The mammaglobin-A DNA vaccine will be administered concurrently with neoadjuvant endocrine therapy, providing a unique opportunity to examine the impact of vaccination in the tumor microenvironment.

Safety and preliminary evidence of biologic efficacy of a mammaglobin-a DNA vaccine in patients with stable metastatic breast cancer.

PURPOSE: Mammaglobin-A (MAM-A) is overexpressed in 40% to 80% of primary breast cancers. We initiated a phase I clinical trial of a MAM-A DNA vaccine to evaluate its safety and biologic efficacy. EXPERIMENTAL DESIGN: Patients with breast cancer with stable metastatic disease were eligible for enrollment. Safety was monitored with clinical and laboratory assessments. The CD8 T-cell response was measured by ELISPOT, flow cytometry, and cytotoxicity assays. Progression-free survival (PFS) was described using the Kaplan-Meier product limit estimator. RESULTS: Fourteen subjects have been treated with the MAM-A DNA vaccine and no significant adverse events have been observed. Eight of 14 subjects were HLA-A2(+), and the CD8 T-cell response to vaccination was studied in detail. Flow cytometry demonstrated a significant increase in the frequency of MAM-A-specific CD8 T cells after vaccination (0.9% ± 0.5% vs. 3.8% ± 1.2%; P < 0.001), and ELISPOT analysis demonstrated an increase in the number of MAM-A-specific IFNγ-secreting T cells (41 ± 32 vs. 215 ± 67 spm; P < 0.001). Although this study was not powered to evaluate progression-free survival (PFS), preliminary evidence suggests that subjects treated with the MAM-A DNA vaccine had improved PFS compared with subjects who met all eligibility criteria, were enrolled in the trial, but were not vaccinated because of HLA phenotype. CONCLUSION: The MAM-A DNA vaccine is safe, capable of eliciting MAM-A-specific CD8 T-cell responses, and preliminary evidence suggests improved PFS. Additional studies are required to define the potential of the MAM-A DNA vaccine for breast cancer prevention and/or therapy.

Targeted pancreatic cancer therapy with the small molecule drug conjugate SW IV-134.

Pancreatic adenocarcinoma is highly resistant to conventional therapeutics and has been shown to evade apoptosis by deregulation of the X-linked and cellular inhibitors of apoptosis proteins (XIAP and cIAP). Second mitochondria-derived activator of caspases (Smac) induces and amplifies cell death by reversing the anti-apoptotic activity of IAPs. Thus, Smac-derived peptide analogues (peptidomimetics) have been developed and shown to represent promising cancer therapeutics. Sigma-2 receptors are overexpressed in many proliferating tumor cells including pancreatic cancer. Selected ligands to this receptor are rapidly internalized by cancer cells. These characteristics have made the sigma-2 receptor an attractive target for drug delivery because selective delivery to cancer cells has the potential to increase therapeutic efficacy while minimizing toxicity to normal tissues. Here, we describe the initial characterization of SW IV-134, a chemically linked drug conjugate between the sigma-2 ligand SW43 and the Smac mimetic SW IV-52 as a novel treatment option for pancreatic adenocarcinoma. The tumor killing characteristics of our dual-domain therapeutic SW IV-134 was far greater than either component in isolation or in an equimolar mix and suggests enhanced cellular delivery when chemically linked to the sigma-2 ligand. One of the key findings was that SW IV-134 retained target selectivity of the Smac cargo with the involvement of the NF-κB/TNFα signaling pathway. Importantly, SW IV-134 slowed tumor growth and improved survival in murine models of pancreatic cancer. Our data support further study of this novel therapeutic and this drug delivery strategy because it may eventually benefit patients with pancreatic cancer.

Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3.

BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies.

A phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma.

PURPOSE: This phase I study was conducted to determine the safety profile and maximum tolerated dose (MTD) of IMP321, a soluble lymphocyte activation gene-3 (LAG-3) Ig fusion protein and MHC Class II agonist, combined with gemcitabine in patients with advanced pancreatic adenocarcinoma. PATIENTS AND METHODS: Patients with advanced pancreatic adenocarcinoma were treated with gemcitabine (1,000 mg/m(2))(level 1), gemcitabine (1,000 mg/m(2)) plus IMP 321 at 0.5 mg (level 2) and 2.0 mg (level 3), respectively. Safety, toxicity, and immunological markers at baseline and post treatment were assessed. RESULTS: A total of 18 patients were enrolled to the study, and 17 were evaluable for toxicity. None of the 6 patients who received 0.5 mg IMP321 experienced IMP321-related adverse events. Of the 5 patients who received IMP321 at the 2 mg dose level, 1 experienced rash, 1 reported hot flashes and 2 had mild pain at the injection sites. No severe adverse events previously attributed to IMP321 were observed. No significant differences were observed when comparing pre- and post-treatment levels of monocytes (CD11b+CD14+), conventional dendritic cells (CD11c+) or T cell subsets (CD4, CD8). CONCLUSIONS: IMP321 in combination with gemcitabine is a well-tolerated regimen. IMP321 did not result in any severe adverse events. No incremental activity observed for the additional IMP 321 to gemcitabine at the dose levels evaluated, likely due to sub-optimal dosing. Immunological markers suggested that higher dose levels of IMP321 are needed for future clinical studies.

Mammaglobin-A cDNA vaccination of breast cancer patients induces antigen-specific cytotoxic CD4+ICOShi T cells.

Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A-specific CD8 T cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this article, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage-IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28, and 56, and immune responses were assessed at serial time points following vaccination. At 6 months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOS(hi) T cells from 5 ± 2 % pre-vaccination to 23 ± 4 % (p < 0.001), with a concomitant decrease in the frequency of CD4+FoxP3+ T cells (regulatory T cells [Treg]) from 19 ± 6 to 10 ± 5 % (p < 0.05). ELISpot analysis of CD4+ICOS(hi) sorted T cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p < 0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p < 0.001). The ratio of CD4+ICOS(hi) T cells to CD4+FoxP3+ T cells increased from 0.37 ± 0.12 before vaccination to 2.3 ± 0.72 (p = 0.021) following vaccination. Further, these activated CD4+ICOS(hi) T cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that Mam-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOS(hi) T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.

Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses.

Tumor-infiltrating immune cells can promote chemoresistance and metastatic spread in aggressive tumors. Consequently, the type and quality of immune responses present in the neoplastic stroma are highly predictive of patient outcome in several cancer types. In addition to host immune responses, intrinsic tumor cell activities that mimic stem cell properties have been linked to chemoresistance, metastatic dissemination, and the induction of immune suppression. Cancer stem cells are far from a static cell population; rather, their presence seems to be controlled by highly dynamic processes that are dependent on cues from the tumor stroma. However, the impact immune responses have on tumor stem cell differentiation or expansion is not well understood. In this study, we show that targeting tumor-infiltrating macrophages (TAM) and inflammatory monocytes by inhibiting either the myeloid cell receptors colony-stimulating factor-1 receptor (CSF1R) or chemokine (C-C motif) receptor 2 (CCR2) decreases the number of tumor-initiating cells (TIC) in pancreatic tumors. Targeting CCR2 or CSF1R improves chemotherapeutic efficacy, inhibits metastasis, and increases antitumor T-cell responses. Tumor-educated macrophages also directly enhanced the tumor-initiating capacity of pancreatic tumor cells by activating the transcription factor STAT3, thereby facilitating macrophage-mediated suppression of CD8(+) T lymphocytes. Together, our findings show how targeting TAMs can effectively overcome therapeutic resistance mediated by TICs.

Therapeutic targeting of pancreatic cancer utilizing sigma-2 ligands.

One major barrier in the development of pancreas cancer therapeutics is the selective delivery of the drugs to their cellular targets. We have previously developed several sigma-2 ligands and reported the discovery of a component of the receptor for these ligands. Several sigma-2 ligands have been shown to trigger apoptosis in pancreas cancer cells. More importantly, sigma-2 ligands are internalized rapidly by the cancer cells and are capable of delivering other small-molecule therapeutics. Here we review sigma-2 ligands and conjugates as a potential novel therapy suitable for investigation in patients with pancreatic cancer.

Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination.

Activation of naïve cluster of differentiation (CD)8(+) cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8(+) T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8(+) T cells and requires CD8α(+)/CD103(+) DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8(+) T-cell responses to viral infection, tumors, and vaccines.

Lysosomal membrane permeabilization is an early event in Sigma-2 receptor ligand mediated cell death in pancreatic cancer.

BACKGROUND: Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. RESULTS: Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. CONCLUSIONS: Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.

Regulating mammalian target of rapamycin to tune vaccination-induced CD8(+) T cell responses for tumor immunity.

Vaccine strategies aimed at generating CD8(+) T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8(+) T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8(+) T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8(+) T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8(+) T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8(+) T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

Pancreatic adenocarcinoma induces bone marrow mobilization of myeloid-derived suppressor cells which promote primary tumor growth.

PURPOSE: Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA). EXPERIMENTAL DESIGN: Peripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15(+)CD11b(+)) by flow cytometry and compared to cancer-free controls. The suppressive capacity of MDSC (CD11b(+)Gr-1(+)) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1(+)CD11b(+)), effector T cells, and tumor cytokine levels. RESULTS: Patients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8(+) T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10. CONCLUSIONS: MDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.

Use of multifunctional sigma-2 receptor ligand conjugates to trigger cancer-selective cell death signaling.

One major challenge in the development of cancer therapeutics is the selective delivery of the drugs to their cellular targets. In the case of pancreatic cancer, the σ-2 receptor is a unique target that triggers apoptosis upon activation. We have previously developed a series of chemical compounds with high affinity for the σ-2 receptor and showed rapid internalization of the ligands. One particular specific ligand of the σ-2 receptor, SV119, binds to pancreatic cancer cells and induces target cell death in vitro and in vivo. In this study, we characterized the ability of SV119 to selectively deliver other death-inducing cargos to augment the cytotoxic properties of SV119 itself. When conjugated to SV119, small molecules that are known to interfere with intracellular prosurvival pathways retained their ability to induce cell death, the efficiency of which was enhanced by the combinatorial effect of SV119 delivered with its small molecule cargo. Our findings define a simple platform technology to increase the tumor-selective delivery of small molecule therapeutics via σ-2 ligands, permitting chemotherapeutic synergy that can optimize efficacy and patient benefit.

Potential targets for pancreatic cancer immunotherapeutics.

Pancreatic adenocarcinoma is the fourth leading cause of cancer death with an overall 5-year survival of less than 5%. As there is ample evidence that pancreatic adenocarcinomas elicit antitumor immune responses, identification of pancreatic cancer-associated antigens has spurred the development of vaccination-based strategies for treatment. While promising results have been observed in animal tumor models, most clinical studies have found only limited success. As most trials were performed in patients with advanced pancreatic cancer, the contribution of immune suppressor mechanisms should be taken into account. In this article, we detail recent work in tumor antigen vaccination and the recently identified mechanisms of immune suppression in pancreatic cancer. We offer our perspective on how to increase the clinical efficacy of vaccines for pancreatic cancer.

Cancer genome sequencing and its implications for personalized cancer vaccines.

New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.

The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine.

BACKGROUND: Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy. RESULTS: The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities. CONCLUSIONS: This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.

Induction of Th17 cells in the tumor microenvironment improves survival in a murine model of pancreatic cancer.

An important mechanism by which pancreatic cancer avoids antitumor immunity is by recruiting regulatory T cells (Tregs) to the tumor microenvironment. Recent studies suggest that suppressor Tregs and effector Th17 cells share a common lineage and differentiate based on the presence of certain cytokines in the microenvironment. Because IL-6 in the presence of TGF-ß has been shown to inhibit Treg development and induce Th17 cells, we hypothesized that altering the tumor cytokine environment could induce Th17 and reverse tumor-associated immune suppression. Pan02 murine pancreatic tumor cells that secrete TGF-ß were transduced with the gene encoding IL-6. C57BL/6 mice were injected s.c. with wild-type (WT), empty vector (EV), or IL-6-transduced Pan02 cells (IL-6 Pan02) to investigate the impact of IL-6 secretion in the tumor microenvironment. Mice bearing IL-6 Pan02 tumors demonstrated significant delay in tumor growth and better overall median survival compared with mice bearing WT or EV Pan02 tumors. Immunohistochemical analysis demonstrated an increase in Th17 cells (CD4(+)IL-23R(+) cells and CD4(+)IL-17(+) cells) in tumors of the IL-6 Pan02 group compared with WT or EV Pan02 tumors. The upregulation of IL-17-secreting CD4(+) tumor-infiltrating lymphocytes was substantiated at the cellular level by flow cytometry and ELISPOT assay and mRNA level for retinoic acid-related orphan receptor γt and IL-23R by RT-PCR. Thus, the addition of IL-6 to the tumor microenvironment skews the balance toward Th17 cells in a murine model of pancreatic cancer. The delayed tumor growth and improved survival suggests that induction of Th17 in the tumor microenvironment produces an antitumor effect.

A genetically encoded multifunctional TRAIL trimer facilitates cell-specific targeting and tumor cell killing.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L) has been shown to exhibit potent and specific apoptotic activity against tumor cells. Several TRAIL constructs have been tried in patients, and the molecule remains under active clinical investigation. Native and recombinant TRAIL must form a homotrimer to become biologically active. However, noncovalently associated TRAIL displays a high degree of sensitivity to degradation, which limits its therapeutic potential. To enforce trimerization of the recombinant protein, we developed a covalently linked TRAIL trimer (TR3) by genetic fusion. This molecular drug design conferred improved stability without altering the native killing ability of TRAIL. Target specificity was shown by blocking TR3 activity with soluble death receptor 5 (DR5-Fc). In addition, we have shown that TR3 is amenable to further, genetic modifications. The incorporation of additional functional domains to TR3, such as antibody fragments (scFvs) that allow for a more cell-specific delivery of the agent, is stoichiometrically controlled and inconsequential with regard to the bioactivity of TRAIL. As proof of this concept, TR3 activity was targeted to the mouse RBC membrane. TR3-decorated RBCs were effectively capable of target cell killing in a model of pancreatic cancer. TR3 represents a generally applicable platform tool to study basic mechanisms along the death receptor pathway. More importantly, the ability to target TR3 to a cell surface presents the opportunity to create a cancer-selective drug with fewer off-target toxicities and enhanced killing capacities.