Carl Ruby

Current status of granulocyte–macrophage colony-stimulating factor in the immunotherapy of melanoma

In 2012, it was estimated that 9180 people in the United States would die from melanoma and that more than 76,000 new cases would be diagnosed. Surgical resection is effective for early-stage melanoma, but outcomes are poor for patients with advanced disease. Expression of tumor-associated antigens by melanoma cells makes the disease a promising candidate for immunotherapy. The hematopoietic cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) has a variety of effects on the immune system including activation of T cells and maturation of dendritic cells, as well as an ability to promote humoral and cell-mediated responses. Given its immunobiology, there has been interest in strategies incorporating GM-CSF in the treatment of melanoma. Preclinical studies with GM-CSF have suggested that it has antitumor activity against melanoma and can enhance the activity of anti-melanoma vaccines. Numerous clinical studies have evaluated recombinant GM-CSF as a monotherapy, as adjuvant with or without cancer vaccines, or in combination with chemotherapy. Although there have been suggestions of clinical benefit in some studies, results have been inconsistent. More recently, novel approaches incorporating GM-CSF in the treatment of melanoma have been evaluated. These have included oncolytic immunotherapy with the GM-CSF–expressing engineered herpes simplex virus talimogene laherparepvec and administration of GM-CSF in combination with ipilimumab, both of which have improved patient outcomes in phase 3 studies. This review describes the diverse body of preclinical and clinical evidence regarding use of GM-CSF in the treatment of melanoma.

Pseudomonas aeruginosa Exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines.

In patients with malignancy, the major barrier to achieving complete response is emergence of resistance to current chemotherapeutic agents. One of the major mechanisms by which tumour cells become resistant to therapies is by altering cellular drug targets through mutations and/or deletions. Resistance by this mechanism is achieved more easily if the drug has limited cellular targets and/or processes. We hypothesized that as Pseudomonas aeruginosa exotoxin T (ExoT) targets six proteins that are required for cancer cell survival and proliferation, it is highly unlikely for cancer cells to develop resistance to this toxin. We assessed ExoTs cytotoxicity against multiple invasive and highly resistant tumour cell lines in order to evaluate its potential as a chemotherapeutic agent. Our data demonstrated that ExoT induced potent cytotoxicity in all tumour cell lines that we examined. Collectively, our data highlighted the potential of ExoT as a possible chemotherapeutic candidate for the treatment of cancer.

Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. Invitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase.

TroVax® vaccine therapy for renal cell carcinoma

Renal cell carcinoma (RCC) is the most common primary malignancy affecting the kidney. In the past decade, several well-designed clinical trials have shifted the treatment paradigm for RCC to favor targeted therapies as first-line agents. Recognition of the immunogenic nature of RCC has also resulted in the development of immunotherapy approaches with high-dose IL-2 treatment being the best established and associated with durable disease control. The lack of defined antigens in RCC has hindered more specific vaccine development. TroVax(®) is a novel vaccine based on a modified vaccinia virus Ankara vector engineered to express the 5T4 tumor-associated antigen, found on over 95% of clear cell and papillary RCC tumors. The safety and efficacy of TroVax has been evaluated in several Phase I/II clinical trials and in a multicenter Phase III trial. This article will discuss the clinical background of RCC, the rationale for TroVax development, results of several TroVax clinical trials and future directions for optimizing TroVax therapy in patients with RCC and other cancers.

Oncolytic virus therapy for cancer

The use of oncolytic viruses to treat cancer is based on the selection of tropic tumor viruses or the generation of replication selective vectors that can either directly kill infected tumor cells or increase their susceptibility to cell death and apoptosis through additional exposure to radiation or chemotherapy. In addition, viral vectors can be modified to promote more potent tumor cell death, improve the toxicity profile, and/or generate host antitumor immunity. A variety of viruses have been developed as oncolytic therapeutics, including adenovirus, vaccinia virus, herpesvirus, coxsackie A virus, Newcastle disease virus, and reovirus. The clinical development of oncolytic viral therapy has accelerated in the last few years, with several vectors entering clinical trials for a variety of cancers. In this review, current strategies to optimize the therapeutic effectiveness and safety of the major oncolytic viruses are discussed, and a summary of current clinical trials is provided. Further investigation is needed to characterize better the clinical impact of oncolytic viruses, but there are increasing data demonstrating the potential promise of this approach for the treatment of human and animal cancers.

Interleukin-2 alters distribution of CD144 (VE-cadherin) in endothelial cells

BackgroundHigh-dose IL-2 (HDIL2) is approved for the treatment of metastatic melanoma and renal cell carcinoma, but its use is limited in part by toxicity related to the development of vascular leak syndrome (VLS). Therefore, an understanding of the mechanisms that underlie the initiation and progression of HDIL2-induced increases in endothelial cell (EC) permeability leading to VLS are of clinical importance.MethodsWe established a novel ex vivo approach utilizing primary human pulmonary microvascular ECs to evaluate EC barrier dysfunction in response to IL-2.ResultsComplementary in vitro studies using exogenous IL-2 and ex vivo studies using serum from patients treated with IL-2 demonstrate that HDIL2 induces VLS through CD144 (vascular endothelial (VE)-cadherin) redistribution.ConclusionsThese findings provide new insight into how IL-2 induces VLS and identifies VE-cadherin as a potential target for preventing IL-2-related VLS.

Early-onset age-related changes in dendritic cell subsets can impair antigen- specific T helper 1 (Th1) CD4 T cell priming

Decline in CD4 T cell immune responses is associated with aging. Although a number of immunological defects have been identified in elderly mice (>18 months old), a key early‐onset immune defect at middle age could be a driver or contributor to defective CD4 T cell responses. Our studies demonstrate that age‐related alterations in DC subsets within the priming environment of middle‐aged mice (12 months old) correlate with and can directly contribute to decreases in antigen‐specific CD4 T cell Th1 differentiation, which measured by T‐bet and IFN‐γ expression, was decreased significantly in T cells following VSV infection or s.c. immunization with a protein antigen in the context of immune stimulation via OX40. The deficient Th1 phenotype, observed following protein antigen challenge, was found to be the result of an age‐related decrease in an inflammatory DC subset (CD11b+ Gr‐1/Ly6C+) in the dLN that corresponded with T cell dysfunction. In the virus model, we observed significant changes in two DC subsets: mDCs and pDCs. Thus, different, early age‐related changes in the DC profile in the priming environment can significantly contribute to impaired Th1 differentiation, depending on the type of immunological challenge.

Combination immunotherapy with anti-CTLA-4 and interleukin-2 redirects regulatory T cells into tumor-draining lymph nodes and expands anti-tumor CD8+ T cells in the tumor microenvironment

Meeting abstracts Monotherapy with Ipilimumab (anti-CTLA-4 antibody) and monotherapy with IL-2 (T cell stimulating cytokine) are approved for the treatment of metastatic melanoma. Combination immunotherapy has been suggested as a more potent regimen but has not been sufficiently investigated. We

The impact of age on a mitoxantrone-based tumor vaccine.

Several chemotherapies, including the anthracycline analog mitoxantrone, are known to induce an immunogenic cell death in tumor cells that can produce a protective anti-tumor immune response in mice. Although the anti-tumor immune response mediated by mitoxantrone tumor cell death has been well described, it is not fully understood what effect age has on these responses. Our objectives were to 1) determine if older mice (12 month old) produced a protective immune response following immunization with mitoxantrone-treated CT26 colon carcinoma cells and 2) determine the age-related cellular and molecular deficiencies that could affect the generation of the protective immune response. CT26 cells treated in vitro with mitoxantrone (1.0 μM) and injected s.c. into an immune competent two-month old mouse resulted in significant protection from a secondary tumor challenge with untreated CT26 (~75% survival). However, older mice failed to generate a protective immune response and succumbed to the secondary tumor challenge (0% survival). To determine the age-related cellular and molecular dysfunction, vaccine-draining lymph nodes (vdLNs) and the vaccination site were harvested and immune cells assessed. There was a significant age-related change in the frequency of several dendritic cell subsets in the vdLNs, as older mice experienced a decrease in both CD8+ and plasmacytoid DC subsets compared to young controls. Alterations in these DC subsets could limit the generation of a protective immune response. These findings demonstrate that older subjects may not generate fully protective anti-tumor immune responses following mitoxantrone chemotherapy.

Combination immunotherapy with Interleukin-2 and CTLA-4 blockade decreases tumor growth and improves overall survival

Background Combination immunotherapy is quickly gaining attention in the field of cancer treatment. IL-2, a cytokine which activates T cells, and ipilimumab, a monoclonal antibody that blocks CTLA-4, are both approved as monotherapy for metastatic melanoma. To date, combination immu- notherapy with IL-2 and anti-CTLA-4 has not been adequately investigated. We hypothesized that that this combination may work synergistically owing to its dis- tinct but complementary mechanisms of T cell activation. mean tumor area with combination IL-2 and anti-CTLA- 4 was 2mm2, while in the anti-CTLA-4 only, IL-2 only, and placebo groups it was 14, 29, and 68 mm2, respec- tively (P<0.01 for all comparisons, except IL-2 only ver- sus anti-CTLA-4 only (not significant)). At day 30, the overall survival with combination IL-2 and anti-CTLA-4 was 50%, while with IL-2 only and anti-CTLA-4 only it was 10% and 20%, respectively (P<0.01 for all compari- sons). All animals treated in the placebo group suc- cumbed to their tumors by day 19. These findings were confirmed in a second experiment with similar results. Conclusions Combination immunotherapy with IL-2 activation and CTLA-4 blockade significantly decreases tumor growth and increases overall survival when compared to IL-2 or anti-CTLA-4 monotherapy or placebo. The role of CD8+ effector versus CD4+ regulatory T cells in the success of this immunotherapy is ongoing and will be included in our formal presentation. Ultimately, we aim to translate this work into a combination immunotherapy clinical trial.