pbi-shRNA™ Furin DNA Plasmid) Vaccine in Advanced Melanoma

CANCER - IMMUNOTHERAPY I 199. Phase II Study of FANG™ (Autologous Tumor Cell – GMCSF/pbi-shRNA™ Furin DNA Plasmid) Vaccine in Advanced Melanoma

John Nemunaitis,1,2,3,4 Neil Senzer,1,4 Minal Barve,3 Padmasini Kumar,4 Donald D. Rao,4 Gladice Wallraven,4 Beena O. Pappen,4 Joseph Kuhn,5 Peter Beitsch,2 Robert Steckler,2 Brian M. Gogel,2 Walton Taylor,2 Phillip B. Maples.4 1 Mary Crowley Cancer Research Centers, Dallas; 2Medical City HCA, Dallas; 3Texas Oncology, P.A., Dallas; 4Gradalis, Inc., Dallas; 5WLS Surgical Associates, P.A., Dallas. The Phase I trial of FANG™ vaccine (Senzer et al Mol Ther 2012; 20:679-686) provided sufficient safety data to proceed to Phase II efficacy studies. This decision was bolstered by the correlation of duration of survival with induced immune response at  3 months (in patients with serial analysis). Specifically, 4 metastatic melanoma patients in the Phase I trial, who having failed standard therapy had an expected survival prognosis of 6 months, received a combined total of 25 vaccinations (i.e. 3 patients at dose level 1 x 107 and 1 patient at 2.5 x 107 cells / injection (database review revealed no evidence of dose related response)). These patients have thus far experienced a median survival of 698 days since starting treatment (967, 835, 560, and 490 days, each). Two of these patients demonstrated induced T cell activation via IFN gamma ELISPOT assessment by Month 3 post treatment start. One patient did not show positive T cell activation at the Month 3 ELISPOT assessment but did so by Month 7. One of the patients who, coming off treatment early and not having a Month 3 assessment, was ELISPOT positive at Month 16. These 4 patients also showed knockdown of the endogenous immunosuppressive proteins TGF1 (mean 99% knockdown) and TGF2 (mean 86% knockdown) mediated by targeted pbi-shRNA™ furin transfected vaccine product. Based on these results, the Phase II trial (CL-PTL 114, BB-IND 14205) was activated following FDA, RAC, IBC and IRB acceptance. Design of the ongoing Phase II study involves an intradermal injection of the FANG™ vaccine (1.0 x 107 cells/injection/ month; maximum of 12 vaccinations). Four patients with Stage IV melanoma with biopsy accessible lesions have been treated. Neither hematologic function, liver enzyme, renal function, or electrolyte assays nor protocol specified physical examinations demonstrated any toxic effects. Immune function analyses, including intratumoral immune phenotype and ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens, are ongoing and will be reported. Stable disease has been observed in 2 of 4 treated patients. In conclusion, results support continuation of accrual.

200. Renal Cell Tumor-Mediated Reprogramming of Natural Killer Cells by Soluble Immune Modulators

Yue Guan,1 Purba Singh,1 Trenae Mann,1 Christopher B. Chambers,1 Donald S. Torry,1 Andrew Wilber.1 1 Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL.

Natural killer (NK) cells classically are associated with immune surveillance and destruction of tumor cells via cytotoxicity. Recent studies indicate that NK cells with non-classical CD phenotypes (CD56brightCD16dim-negative) lose their cytotoxic capacity, release pro-angiogenic factors, and facilitate tumor growth. Mechanisms driving these changes are not established but soluble factors are thought to direct the unique phenotypic and functional differentiation of these CD56brightCD16- NK cells. We used a combination of clinical specimens, primary cell culture, and a novel animal model to evaluate the effects of transforming growth factor beta (TGF) and prostaglandin E2 (PGE2) on NK cell phenotype and function in renal cell carcinoma (RCC). Plasma levels of PGE2 for RCC patients (N=7) were 92 + 39 pg/mL before surgery and 45 + 11 pg/ S78

mL after nephrectomy (P=0.02, T-test). Post-operative levels of PGE2 were approaching levels observed for healthy volunteers (27 + 11 pg/mL), suggesting that PGE2 is a RCC tumor-derived factor. Phenotype analysis revealed that peripheral blood NK (pNK) cells from healthy volunteers were characteristically CD56dimCD16bright (95%); however, RCC tumor-derived NK cells exhibited the CD56brightCD16- phenotype. The tumor-derived NK cells had elevated vascular endothelial growth factor (VEGF) transcripts (3fold) compared to freshly isolated pNK cells. Trans-differentiation of CD56dimCD16bright pNK cells to the CD56brightCD16dim phenotype was achieved after culture with TGF; this conversion was coincident with impaired NK cytotoxicity (>50% reduction) and a modest augmentation of VEGF mRNA upon crosslinking of the activating receptor NKp46. A Balb/c mouse renal adenocarcinoma cell line (Renca) expressing moderate levels of TGF (300-350 pg/mL) was engineered for PGE2 expression with murine Cox2 in antisense (Cox2-) or sense (Cox2+) orientations. ELISA demonstrated that Cox2- cells produced background levels of PGE2 (35 pg/mL) while Cox2+ cells produced >5,000 pg/mL PGE2. Exposure of freshly isolated human pNK cells to conditioned supernatants from Cox2(TGF only) or Cox2+ (TGF and PGE2) cells reduced cytotoxicity by 20% and 30%, respectively. In vivo studies demonstrated rapid tumor growth and metastasis over three weeks following kidney capsule injection in Balb/c mice. Lymphocytes isolated from these RCC-like tumors were infiltrated with NK-cells (CD3-p46+), nearly 60% of which demonstrated diminution or absence of CD16. Collectively, these studies support a role for TGF and PGE2 in trans-differentiation of CD56dimCD16bright pNK cells to CD56brightCD16dim-negative cells which have lost their normal destructive capacities and, instead, support tumor growth and metastasis. Studies designed to inhibit the immune suppressive functions of these molecules are planned and may provide new therapeutic avenues for future cancer treatments.

201. A New Epithelial Junction Opener for Cancer Therapy

Ines Beyer,1 Hua Cao,1 Jonas Persson,1 Roma Yumul,1 Andre Lieber.1 1 Medicine, University of Washington, Seattle. Most solid tumors are of epithelial origin. Epithelial cancers maintain intercellular junctions that link cancer cells together and prevent the penetration of therapeutics deep into the cancer. Several studies demonstrated that the upregulation of epithelial junction proteins correlated with increased resistance to therapy, including therapy with monoclonal antibodies and chemotherapeutics. One of the epithelial junction proteins is desmoglein 2 (DSG2). Recently, we demonstrated that a group of human adenoviruses (Ad) (Ad serotype 3, 7, 11, and 14) use DSG2 as a primary attachment receptor for the infection of cells. DSG2 is overexpressed in epithelial malignancies. We have created a small recombinant protein derived from Ad serotype 3. This protein binds to DSG2 and triggers transient opening of epithelial junctions. We therefore named the protein “JO-1” (“junction opener -1”). JO-1 is produced in E.coli and can be easily purified. In a number of xenograft tumor models, we have shown that intravenous injection of JO-1 increased the efficacy of monoclonal antibody (e.g. trastuzumab/Herceptin and cetuximab/ Erbitux) therapy. Many chemotherapy drugs are larger than 500 Da, the upper limit of molecules that are able to pass through tight junctions. We have demonstrated that the therapeutic effects of several of these drugs, including paclitaxel, nab-paclitaxel, docetaxel, irinotecan, and liposomal doxorubicin are increased by co-therapy with JO-1. Furthermore, we have shown that JO-1 leads to massive accumulation of chemotherapy drugs in the tumor, decreasing the drug concentrations in the rest of the body. This resulted in the elimination of chemotherapy-related toxicities to bone marrow, liver, and intestine. Toxicology studies carried out in mice and monkeys have Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy